Introduction

Corynebacterium glutamicum is a Gram-positive, non-sporulating, and facultatively anaerobic bacterium (Kinoshita 1985; Yukawa et al. 2006; Nishimura et al. 2008) that has been used in chemical production such as lactate, glutamate, and lysine (Kinoshita 1985; Malumbers et al. 1995; Inui et al. 2004a, b; Okino et al. 2005). The fermentation conditions for mass production methods using this bacterial species are well-established (Kikuchi et al. 2008; Liebl and Sinskey 1990). However studies on protein secretions in C. glutamicum are only now starting to gain traction since the microorganism has long been considered able to excrete only a limited number of proteins in its culture medium. This view should change with the realization that C. glutamicum harbors much more protein secretion potential than previously thought (Watanabe et al. 2009). Moreover, its lack of detectable extracellular hydrolytic enzyme activity (Billman-Jacobe et al. 1995) makes C. glutamicum a very favorable and versatile host for heterologous protein productions, capable of much more protein secretion than the dozen or so documented to date (Billman-Jacobe et al. 1995; Date et al. 2003, 2004, 2006; Itaya and Kikuchi 2008; Kikuchi et al. 2006; Liebl et al. 1992; Meissner et al. 2007; Salim et al. 1997; Smith et al. 1986). C. glutamicum naturally secretes two major proteins of which PS2 is more strongly secreted than PS1 (Peyret et al. 1993). The PS2 secretion signal is therefore more popular in current extracytoplasmic production, but it has the major drawback that PS2-based protein secretion yields widely vary depending on target proteins (Kikuchi et al. 2003; Watanabe et al. 2009). This drawback has meant that overall application of C. glutamicum as a host in protein secretion remains comparatively limited.

Of 108 active signal sequences of C. glutamicum, that of CgR0949 is particularly potent, exceeding the strength of the PS2 signal by more than two orders of magnitude (Watanabe et al. 2009). Being a Tat-type sequence, CgR0949 may transport a wide variety of pre-folded proteins, including cofactor requiring redox enzymes, multimeric proteins, and membrane proteins, across the cell membrane (Lee et al. 2006). It should therefore easily find use in protein productions in bioindustry if the current problem of significantly lower yields of Tat-exported proteins when compared to the 1 g l−1 yields of Sec-exported proteins (Lee et al. 2006) can be solved. Further fine tuning for the expression of Tat-type secreted protein, cell physiology, and fermentation conditions is required. In this study, an efficient Tat-type signal sequence encoded on cgR_0949 ORF of C. glutamicum R in conjunction with optimized fermentation conditions and gene expression system enabled secretion of up to 1.8 g l−1 active green fluorescent protein (GFP) in culture medium. This is the first report showing high-level secretion of a heterologous protein in C. glutamicum using its own signal sequence.

Materials and methods

Bacterial strains and culture conditions

The bacterial strains used in this study are summarized in Table 1. E. coli was cultivated in Luria–Bartani (LB) medium at 37°C with vigorous shaking (Sambrook et al. 1989). C. glutamicum R was grown at 33°C in A medium [2 g yeast extract, 7 g casamino acids, 2 g urea, 7 g (NH4)2SO4, 0.5 g KH2PO4, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 6 mg FeSO4·7H2O, 4.2 mg MnSO4·H2O, 0.2 mg biotin, and 0.2 mg thiamine per 1 l] supplemented with 4% glucose on a rotary shaker at 200 rpm. Chloramphenicol (Wako Pure Chemical, Osaka, Japan) was used at the following concentrations: for E. coli, 50 μg ml−1 and for C. glutamicum, 5 μg ml−1.

Table 1 Bacterial strains and plasmids used in this study
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DNA manipulations

E. coli plasmid DNA was isolated using QIAprep spin kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. E. coli was transformed by the CaCl2 method (Sambrook et al. 1989). In the case of C. glutamicum plasmid extraction, cells were treated with 4 mg lysozyme/ml at 37°C for 30 min. Restriction endonucleases were purchased from TAKARA BIO Inc. (Shiga, Japan). DNA sequencing was performed on an ABI PRISM 3130xl genetic analyzer with a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. DNA sequence data were analyzed using Genetyx WIN program (Genetyx, Tokyo, Japan).

Construction of plasmids for GFP production

The green fluorescence protein (GFP), AcGFP1 (27 kDa) from Aequorea coerulescens was used as a marker. To express and secrete GFP efficiently in C. glutamicum, four plasmids of which the upstream regions of the GFP gene were different from were constructed. First, a DNA fragment containing GFP gene, gfp was amplified from pAcGFP1 vector (Clontech Laboratories, Mountain View, CA, USA) by PCR with primers 1 and 2 (Table 2). The fragment was digested with XhoI and SphI and ligated to the same site of pCRC900 (Table 1) to replace α-amylase gene. pCRC900 can express a gene using tac promoter in C. glutamicum. The extracted plasmid was designated pCRC901 (Table 1). DNA fragment encoding CgR0949 signal sequence was amplified by PCR with primers 3 and 4 (Table 2), purified by gel electrophoresis and extraction, digested with EcoRV and ligated to the same site of pCRC901 using ligation high (TOYOBO) (pCRD313).

Table 2 Oligonucleotides used in this study
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Second, to change the Shine–Dalgarno sequence for GFP expression, the DNA fragment encoding a chimeric protein of CgR0949 and GFP was further amplified by PCR with primers 5 and 6 from pCRD313. The fragment was digested with MunI and BamHI and ligated to EcoRI and BamHI site of pCRD310, which has the Shine–Dalgarno sequence of triosephosphate isomerase derived from C. glutamicum (pCRD314). Third and fourth, plasmids expressing GFP gene containing promoter sequences of cspA (pCRD315) and cspB (pCRD316) from C. glutamicum R were also constructed, respectively (Table 1). The cspA/cspB promoter and CgR0949 signal sequences were fused by crossover PCR. Promoter regions of cspA and cspB extracted from C. glutamicum R genome were 685 bp (region on genome, 3068081–3067397) and 594 bp (region on genome, 2610432–2609839), respectively. First PCR was performed to amplify each promoter or signal sequence from chromosomal DNA of C. glutamicum R with primers 7–12. Second PCRs were carried out to fuse the DNA fragments obtained from each corresponding first PCR. For construction of pCRD315 (316), seconnd PCR was performed with primers 8 and 9 (or 11). The resultant DNA fragments were digested with XhoI and EcoRV and ligated to the same site of pCRC901. After checking the inserted DNA sequence, the resultant plasmids were transformed into E. coli SCS110, and extracted plasmids were used to transform C. glutamicum by electroporation (Vertès et al. 1993). C. glutamicum cells possessing the plasmid were selected on complex solid medium plates containing 1.5% agar (Becton, Dickinson) and chloramphenicol.

Protein secretion conditions

C. glutamicum was inoculated into 40 ml of A medium in a 500-ml baffle flask and cultivated at 33°C for 16 h with shaking at 200 rpm. Then, 30 ml of culture was inoculated into 300 ml of A medium (without urea) and cells incubated at 33°C with constant agitation of 1,000 rpm in a 1-l jar fermentor. To prevent glucose starvation, glucose was continuously added to the cultures at the final concentration of 6% and totally 80 g was supplemented. To monitor the glucose concentration, an aliquot culture was centrifuged, and the supernatants were analyzed by an enzyme electrode glucose sensor (BF-4, Oji Scientific instruments). Cell growth was monitored by measuring OD610 with a spectrophotometer (DU800, Beckman Coulter, CA, USA). Dissolved oxygen concentration was controlled not to be lower than 3.0 ppm using an oxygen gas generator. The pH was monitored using a pH controller (DT-1023, Biott Co Ltd., Tokyo, Japan) and maintained at 7.2 by supplementing with 5 N ammonia. To examine the effect of divalent cations on protein secretion, CaCl2∙2H2O, MgSO4∙7H2O, MnCl2∙4H2O, or ZnCl2 were added to the cultures to a final concentration of 2.0 g l−1. For detergent treatment, Triton X-100 and Tween 20 were used at final concentrations of 0.02% or 0.1%, respectively.

Preparation of extracellular, cell surface, and cytosolic proteins

Supernatants as extracellular fractions were prepared by centrifugation of the culture samples (10,000×g, 4°C; 10 min). To extract cytosolic protein, cells were harvested by centrifugation (5,000×g, 4°C; 10 min) and pelleted. The cells were then washed once with equal volume of extract buffer (100 mM Tris/HCl, pH 7.5, 20 mM KCl, 20 mM MgCl2, 5 mM MnSO4, and 0.1 mM EDTA) and centrifuged again. Since negligible quantities of proteins were obtained from cell supernatant after the second washing, only the first washing supernatant was utilized for the further experiments (as cell surface fraction). Remaining cell pellets were re-suspended with extract buffer and sonicated using an ultrasonic homogenizer (Astrason model XL2020) in an ice-water bath for three 2-min periods, interrupted by 2-min cooling intervals. Cell debris was removed by centrifugation (10,000×g, 4°C; 30 min). The supernatant was used as cytosolic protein samples for GFP detection. Protein concentrations were measured with a Bio-Rad protein assay kit.

Quantification of secreted proteins

GFP was purified from the culture supernatants by hydrophobic interaction column (HIC) chromatography method using Macro-Prep® methyl HIC support (Bio-Rad, Richmond, CA, USA) according to the manufacturer’s instructions. The purity of the separated GFP was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were visualized with CBB staining and scanned by an Image Scanner (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The scanned images were analyzed with ImageMaster Labscan 3.0 (GE Healthcare). Active GFP was analyzed by native PAGE and visualized by the fluorescence mode of Typhoon TRIO (GE Healthcare), with a 532 nm of excitation and 526SP (short-pass) emission filter. GFP amount and its fluorescence signal in gel were linear correlation in our experimental conditions. No signal was detected from denatured (boiled) samples. The actual GFP amount of each sample was calculated using standard curve (0.2–0.8 μg purified GFP). GFP signal was also directly detected by SpectraMax® M2/M2e Microplate Readers (Molecular Devices Corp., Sunnyvale, CA, USA). Excitation, emission, and cut-off spectra were set at 475, 520, and 515 nm respectively. Secreted α-amylase was analyzed using α-amylase assay kit according to the manufacturer’s protocol (Kikkoman Corporation, Chiba, Japan).

RNA isolation

Total RNA was extracted from C. glutamicum R cells using Qiagen RNeasy Mini kit (Qiagen). Cultures were added to two volumes of an RNAprotect bacteria reagent (Qiagen), incubated for 10 min, centrifuged at 6,000×g for 10 min at room temperature. The pellet was resuspended in RLT buffer containing β-mercaptoethanol (RNeasy mini-kit; Qiagen) to a final concentration of 1 g dry cell weight per liter. A 1-ml sample of this cell suspension was subsequently mixed with 0.5 g of 0.1 mm zircon/silica beads (BioSpec Products, Bartlesville, OK, USA). Cells were mechanically disrupted for eight cycles of 45 s at a speed rating of 6.5 spaced by 5 min resting intervals in a Fastprep FP120 instrument (Qbiogene, Heidelberg, Germany). The resulting mixture was centrifuged for 10 min at 20,000×g. The supernatant was processed using an RNeasy system with DNase on-column treatment according to manufacturer’s instructions for RNA extraction. The purity of isolated RNA samples was analyzed by agarose gel electrophoresis and spectrophotometrically and stored at −80°C until ready for use.

Quantitative reverse transcriptase PCR

Quantitative reverse transcriptase PCR (RT-PCR) was performed using an ABI Prism 7000 sequence detection system (Applied Biosystems) and QuantiTect SYBR Green RT-PCR kit (Qiagen) according to the manufacturer’s instructions. Specific primers 13–16 (Table 2) were designated by Primer Express Software v2.0 (Applied Biosystems). Each PCR reaction consisted of 10 μl 2× QuantiTect SYBR Green RT-PCR Master mix, 0.5 μM forward and reverse primers, 0.1 μl reverse transcriptase, 0.4 μl RNase inhibitor, and 20 ng total RNA in a total volume of 20 μl. PCR parameters were at 50°C for 30 min, 95°C for 15 min, and 45 cycles at 95°C for 15 s and 60°C for 1 min. The absolute quantification C T (threshold cycle) method (Applied Biosystems) was used to quantify relative expression, with a threshold cycle being defined as the cycle at which the reporter fluorescence is distinguishable from the background in the extension phase of PCR reaction. The C T values were computed as the average of duplicates.

Results

Vector optimization enhances GFP secretion by C. glutamicum

In order to confirm conditions for production of GFP as secreted protein, C. glutamicum R was transformed with plasmid pCRD313, encoding signal sequence of C. glutamicum R CgR0949 and GFP on the one hand, and plasmid pCRC901 (identical to pCRD313 except for the absence of signal sequence) on the other hand. A chimeric GFP was expressed under the control of the tac promoter. The transformants were flask-cultured in A medium and secreted GFP was qualitatively detected by native PAGE and measurement of GFP fluorescence. As expected, only transformants bearing pCRD313 revealed the presence of GFP in their culture (Fig. 1). Furthermore, the presence of multiple bands indicated that the GFP was multimeric. Likewise, cytosolic and cell-surface fractions derived from the transformants harboring pCRD313 clearly revealed the presence of GFP, whereas only a background fluorescence was observed in pCRC901 transformants (data not shown). Attempts to enhance the GFP production by simply altering cultivation conditions of the pCRD313 transformants from flask to jar fermentor were unsuccessful, as the final yield of GFP with standard A medium remained low (approximately 20 mg l−1).

Fig. 1
figure 1

GFP secretion by C. glutamicum. GFP secretion was detected by native PAGE analysis. Aliquots of concentrated culture supernatants were loaded on 12.5% polyacrylamide gel. Lane 1 Purified GFP; lane 2 the supernatant of strain R/pCRC901; lane 3 the supernatant of strain R/pCRD313. After electrophoresis, GFP fluorescence was visualized by Typhoon TRIO image analyzer

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The other way to increase the amount of secreted GFP is by optimizing expression vectors. First, the 5′ untranslated region (UTR) region including SD sequence of lacα on pCRD313 was replaced with that of triosephosphate isomerase (tpi) gene in C. glutamicum. The tac promoter system is known to express genes effectively in C. glutamicum (Liebl et al. 1992), but its Shine–Dalgarno (SD) sequence is for E. coli and therefore not optimized for C. glutamicum. We previously found that the SD sequence of tpi showed high translation efficiency in C. glutamicum. Therefore, vector pCRD314 containing tac promoter, SD sequence of tpi, and gfp gene was constructed. Second, tac promoter was replaced with promoters of PS1-encoding cspA or PS2-encoding cspB of C. glutamicum, as enhanced heterologous protein secretion was previously achieved using cspB promoter and cspA signal sequences (Date et al. 2004). Therefore, the vectors, pCRD315 and pCRD316, containing cspA promoter and cspB promoter, respectively, were also constructed.

C. glutamicum R was separately transformed with each of these three vectors and the transformants were cultured in A medium at 33°C for 48 h. The fluorescence of the supernatants was measured by SpectraMax. All transformants showed increased fluorescence intensities compared to that of pCRD313 (Fig. 2). However, since the pCRD314 transformants showed highest fluorescence intensity (2.9 times higher), they were used for further analysis.

Fig. 2
figure 2

GFP secretions using various expression systems via the CgR0949 signal sequence. Cells harboring the GFP expressing plasmid were cultivated for 48 h. Supernatants were subjected to SpectraMax analysis and fluorescence (excitation, 475 nm; emission, 520 nm) was measured. Data represent average values in triplicate experiments

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Calcium ions induce secretion of GFP

Changes to cultivation conditions, including treatment with detergents and addition of metal ions to the growth medium, are known to increase protein secretion in bacteria (Boekema et al. 2007; Kikuchi et al. 2002; Yang et al. 1998). Their effects on protein secretion by pCRD314 C. glutamicum transformants were hence investigated. First, Triton X-100 and Tween 20 at various concentrations were added to flask cultures of the transformants, but no increase in GFP secretions was observed (data not shown). On the contrary, Triton X-100 strongly inhibited cell growth at 0.02% concentration. Second, the effect of the divalent cations Mg2+, Mn2+, Zn2+, and Ca2+ were investigated by monitoring cell growth and protein secretion of appropriately supplemented cultures (Figs. 3 and 4). Growth was not inhibited by the addition of Mg2+, Mn2+, or Ca2+, but was strongly inhibited (95% inhibition) by Zn2+ (Fig. 3). GFP secretion in media was very slightly increased by Mg2+ (1.1-fold) and Mn2+ (1.2-fold); however, the drastic induction of GFP secretion was detected by Ca2+ (over 30-fold; Fig. 4a, Table 3). SDS-PAGE analysis showed that the addition of Ca2+ increased the secretion of several proteins besides GFP, even though the predominant protein induced by Ca2+ addition was GFP (Fig. 4b).

Fig. 3
figure 3

Effect of several divalent cations on cell growth. Cells were incubated for 16 h at 33°C in a flask culture and aliquots were inoculated into a 1-l jar fermentor in the presence or absence of several cations. a R with or without Ca2+ (open or closed diamond, respectively), b R/pCRD314 with or without Ca2+ (open or closed triangle, respectively), c R/pCRD314 with Mn2+, d R/pCRD314 with Mg2+, e R/pCRD314 with Zn2+

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Fig. 4
figure 4

Effect of divalent cations on secreted proteins. a Secreted GFP was detected by native PAGE. Ten-microliter aliquots of each supernatant of R/pCRD314 were loaded on 12.5% poly-acrylamide gel and visualized by Typhoon TRIO image analyzer. b Extracellular proteins of wild-type or R/pCRD314 were analyzed by SDS-PAGE. Five-microliter aliquots of each supernatant were loaded on 12.5% SDS poly-acrylamide gel, and proteins were stained by CBB. Black arrows point to the proteins whose secretion was influenced by addition of Ca2+

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Table 3 Amount of GFP detected in each fraction
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Using A medium supplemented Ca2+, concentration of secreted GFP reached 790 ± 84 mg l−1 after 24-h incubation in a jar fermentor (Table 3). A longer period of cultivation further enhanced GFP accumulation (Fig. 5a, Table 3). The maximum amount of secreted GFP (sum of GFP amount in supernatant and cell surface fractions) reached 1640 ± 193 mg l−1 after 48-h incubation in the presence of Ca2+ (Table 3). In contract, the amount was only 24 ± 3.3 mg l−1 at the same time in the absence of Ca2+ (Table 3). Interestingly, the secretion of entire spectrum of secreted proteins of wild type was not enhanced by Ca2+ addition (total extracellular protein after 24-h incubation in calcium-minus culture; 400 mg l−1, calcium-containing culture; 360 mg l−1). The GFP in cytosolic fraction was slightly increased by Ca2+ addition (Fig. 5b). Total cellular proteins amounts were same with or without Ca2+ (11.5 mg protein/g cell weight after 24 h of incubation).

Fig. 5
figure 5

Time-course analysis of extracellular and cytosolic GFP. Cells were incubated in a jar fermentor at 33°C and the GFPs (GFP signal intensities) were analyzed by SpectraMax analyzer. a Extracellular GFP; b cytosolic GFP. Empty symbols indicate the samples grown with Ca2+, while filled symbols indicate samples grown without Ca2+. Error bars show SD (n = 3)

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Ca2+ addition has no effect on gfp gene expression

To investigate the effect of Ca2+ on the GFP secretion, the transcription levels of gfp were analyzed by quantitative RT-PCR. Total RNAs were extracted from cells harvested at mid-, late log (after 4 h and 9 h of incubation), and stationary phases (after 24 and 48 h of incubation), and the accumulation of messenger RNA (mRNA) was compared. The amount of 16S ribosomal RNA was used as internal standard. The results indicated that gfp transcription levels of the cells supplemented Ca2+ were only 1.6 times higher than those of control during the cultivation period (Fig. 6), despite a tremendous induction of GFP secretion that occurred upon the addition of Ca2+.

Fig. 6
figure 6

GFP mRNA accumulation. Cells were incubated at 33°C in a jar fermentor, and mRNAs were isolated and analyzed by qRT-PCR. Filled and empty circles show GFP/16S rRNA ratio obtained from cells in calcium-sufficient or in calcium-deficient cultures, respectively

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The effect of Ca2+ addition on α-amylase secretion

In order to determine the effect of Ca2+ addition on different secretory proteins, α-amylase-producing C. glutamicum strain was constructed. Using pCRD312 vector, α-amylase was expressed under the control of tac promoter, followed by SD sequence of tpi in C. glutamicum. The transformant was cultivated in a jar fermentor for 48 h, and the α-amylase in the supernatant and cell surface fractions were determined by amylase assay kit. The enzyme activity was found in both supernatant and cell surface fractions, and consequently, the activity of α-amylase in calcium-sufficient culture increased five times over that of calcium-deficient culture (Fig. 7).

Fig. 7
figure 7

Secretion of α-amylase by Ca2+ addition. Strain R/pCRD312 was cultivated in a jar fermentor at 33°C with or without Ca2+. After 48 h, secreted α-amylase activity in culture medium and cell surface was analyzed. Gray and black boxes indicate amylase activity in cell surface and supernatant fraction, respectively

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Discussion

Because C. glutamicum does not to have broad-spectrum proteolytic activity, it has been widely considered to secrete only a limited number of proteins, e.g., PS1 and PS2 (Billman-Jacobe et al. 1995). Recent studies have, however, indicated that C. glutamicum indeed possesses a variety of secretory proteins (Watanabe et al. 2009; Hermann et al. 2001), meriting investigation into whether the bacterium might make be good host for protein production. In this report, enhanced secretion of heterologous proteins was studied using GFP and α-amylase as model secreted proteins. Using the signal sequence of the to-date-uncharacterized CgR0949 and optimizing gene expression and fermentation conditions, the concentration of secreted GFP in the medium reached 1.8 g l−1. This GFP yield is double the 0.9 g l−1 of Streptomyces transglutaminase secreted by C. glutamicum using the signal sequence of PS1 and promoter of PS2 (Date et al. 2004); PS1 and PS2 constitute the two major secreted C. glutamicum proteins (Jollif et al. 1992). It also exceeds the 1 g l−1 yields achieved using Sec-type signal sequences (Lee et al. 2006), demonstrating that enhanced C. glutamicum secretion using Tat-type signals like CgR0949 is feasible. In essence, our secretion system married the signal sequence of CgR0949 to an expression system, which fused E. coli tac promoter to the SD sequence of C. glutamicum triose phosphate isomerase-encoding tpi. Replacement of the 5′ UTR sequence of lac gene with that of tpi contributed significantly to the improved translation efficiency. This makes sense considering that efficient expression of tpi is important for cell viability (Solem et al. 2008).

In bacteria, Sec and Tat pathways constitute the two major systems for protein secretion. By virtue of being the pathway through which most bacterial secretions occur, the Sec pathway is the more-studied and consequently more often employed pathway for heterologous protein secretion. Proteins are secreted through the Sec pathway in an unfolded form (Muller and Klosgen 2005; Tjalsma et al. 2000) and may sometimes fail to fold properly out of plasma membrane. Moreover, some proteins can be secreted through both systems. For example, in E. coli, GFP proteins can heterologously excrete through both Sec and Tat pathways, but the GFP secreted through the Sec pathway is inactive (Feilmeier et al. 2000). In contrast, GFP is always secreted in an active form across the plasma membrane by the Tat system (Thomas et al. 2001). The always active Tat-secreted protein has helped heighten interest in the pathway. In one evaluation of Tat-dependent secretion of GFP in the three Gram-positive bacteria Staphylococcus carnosus, B. subtilis, and C. glutamicum, only C. glutamicum was able to excrete active form GFP into its medium (Meissner et al. 2007), suggesting that not all Tat pathways are the same. Other proteins secreted via the C. glutamicum Tat pathway include isomaltodextranase and protein glutaminase (Kikuchi et al. 2006, 2008). This study demonstrated optimizations in Tat-dependent secretion that improve the attractiveness of C. glutamicum as a host for protein production.

In optimizing fermentation conditions, calcium ions, but not magnesium, manganese, or zinc ions, enhanced GFP secretion. The profile of secreted proteins in the presence of calcium differed from that in its absence, with several extracellular proteins up-regulated while others were down-regulated by Ca2+ addition (Fig. 4b). The overall effect of calcium addition was that it led to a 10% decline in the total extracellular protein pool. Irrespective of this, the amount of active form GFP secreted upon Ca2+ addition was over 50 times greater than that in the absence of calcium (Table 3). In contrast, cytosolic GFP increased only 1.3 times in the presence of calcium (Fig. 5), matching the corresponding differences in mRNA levels (1.6 times higher) upon calcium addition (Fig. 6). This may be explained as most of synthesized GFP being immediately released into the culture medium across the plasma membrane due to increased secretion efficiency upon Ca2+ addition. The increased efficiency may be accompanied by a cellular protection mechanism that degrades any excess GFP. GFP mRNA levels were higher in stationary-phase cultures than log-phase cultures, and their variation in log-phase cultures was very low (Fig. 6). Since tac promoter is constitutively expressed in C. glutamicum (Liebl et al. 1992), the expression of GFP by tac promoter may have little effect during jar fermentation with or without Ca2+.

Secretion of α-amylase in our system led to activities that were over five times higher upon Ca2+ addition (Fig. 7). Kikuchi et al. previously demonstrated that calcium-independent pro-transglutaminase was secreted in medium using PS2 signal sequence, which utilizes Sec pathway, and its activity was up-regulated by the addition of 2.0 g l−1 Ca2+ (Kikuchi et al. 2002). In multicellular organisms, Ca2+ is a well-known factor facilitating exocytotic secretion through recruitment of components involved in vesicular and membrane fusion. In eukaryotic cells, SNARE proteins, the cytoplasmic protein cargo for secretion, also drive Ca2+-triggered membrane fusion in exocytosis (Kesavan et al. 2007). In bacteria, however, this type of Ca2+-mediated secretion has not been reported, yet. It is still difficult to explain the drastic increase of GFP accumulation in calcium-sufficient cultures (Table 3), but at least Ca2+ can induce protein secretions involving in PS2 and CgR0949 signal sequences.

Obviously, we cannot eliminate the possibility of calcium influence on transcriptome of C. glutamicum because it is a well-known signal molecule that triggers a wide variety of cellular events in bacteria (Dominguez 2004). Calcium might affect the expression of genes involved in secretion pathway. Further investigation, such as transcriptomic and proteomic analyses, should further clarify the effects of Ca2+ on protein secretion.

C. glutamicum possesses a variety of cell surface proteins by proteomic analysis (Hansmeier et al. 2006). In our secretory system, GFP was additionally detected in the cell surface fraction, suggesting that a part of secreted GFP was trapped on the cell wall of C. glutamicum (Table 3). The C. glutamicum cell envelope containing the mycolic acid layer is known to act as a permeability barrier (Daffé 2005). Of the total GFP, about 20% was located in the cell surface fractions (Table 3), indicating that improving the permeability of cell envelope is needed to increase protein secretion when using Gram-positive bacteria as a host.

In summary, calcium was an essential component for the mass production of heterologous protein using secretion system in this study. To date, the amount of secreted GFP by signal sequences ranges from 0.1 to 12 mg l−1 (Eiden-Plach et al. 2004; Kjaerulff and Jensen 2005; Su et al. 2004). By optimizing culture conditions as well as plasmids, high level secretion of GFP in C. glutamicum was achieved. To improve the secretion efficiency, further tuning is under investigation.