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Keywords:

  • carbon source;
  • Corynebacterium glutamicum;
  • E. coli–C. glutamicum shuttle vector;
  • inducible promoter;
  • lignocellulolytic enzyme;
  • secretion

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Aims:  To obtain strong, carbon source-inducible promoters useful for industrial applications of Corynebacterium glutamicum.

Methods and Results:  DNA microarray and qRT-PCR enabled identification of the promoters of cgR_2367 (malE1) and cgR_2459 (git1) as strong, maltose- and gluconate-inducible promoters, respectively, in C. glutamicum. Promoter probe assays revealed that in the presence of the inducing sugars, PmalE1 and Pgit1, respectively, facilitated 3·4- and 4·2-fold increased β-galactosidase activities compared to the same activity induced by glucose. In addition, PmalE1 was not functional in Escherichia coli, in which Pgit1 function was repressible, which enabled the cloning of a hitherto ‘difficult-to-clone’ heterologous gene of a lignocellulolytic enzyme, whose secretion was consequently induced by the carbon sources.

Conclusions:  PmalE1 and Pgit1 are strong, carbon source-inducible promoters of C. glutamicum whose characteristics in E. coli are integral to the secretion ability of C. glutamicum to secrete lignocellulolytic enzyme.

Significance and Impact of the Study: Corynebacterium glutamicum, like its counterpart industrial workhorses E. coli and Bacillus subtilis, does exhibit strong, carbon source-inducible promoters, and the functionality of two of which was demonstrated in this study. While this study may be most relevant in the ongoing efforts to establish technologies of the biorefinery, it should also be of interest to general microbiologists exploring the versatility of industrial micro-organisms. In so doing, the study should impact future advances in industrial microbiology.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Corynebacterium glutamicum is a high-GC, Gram-positive industrial bacterium with readily manipulable metabolic properties (Jetten et al. 1994; Kawaguchi et al. 2008). This manipulation frequently necessitates the regulation of heterologous gene expression and/or control of homologous gene expression. Efficient, sugar-inducible promoters such as Plac and Pbad of Escherichia coli (Guzman et al. 1995; Newman and Fuqua 1999) and Pxyl of Bacillus subtilis (Kim et al. 1996) are widely used to optimize gene expression in the respective micro-organisms. For corynebacteria, however, equivalent universally used promoter systems are yet to be developed. Instead, promoters such as Plac and Ptac that are functional in C. glutamicum (Tsuchiya and Morinaga 1988) are often utilized as strong constitutive promoters in C. glutamicum even although in so doing, situations where expression of the target gene is not strong enough occasionally arise (Salim et al. 1997), especially when the gene is integrated in the chromosome.

Another bottleneck in genetic engineering of this bacterium arises when the target gene product has deleterious effects on E. coli cells transformed by E. coli–C. glutamicum shuttle vectors, as cloning of the gene is rendered impractical. To avoid such effects, prior assessment of heterologous functionality of Corynebacterium promoters in E. coli becomes a necessity of shuttle vector construction (Patek et al. 2003a,b).

The complete sequencing of C. glutamicum (Kalinowski et al. 2003; Yukawa et al. 2007) has enabled global analysis of promoter regulation (Hayashi et al. 2002; Polen and Wendisch 2004). The disparate profile of C. glutamicum’s carbon source-regulation promoters (Gerstmeir et al. 2004; Letek et al. 2006) attests to the wide variety of carbon sources which the organism can utilize (Liebl 1991).

To overcome the aforementioned drawback both to the development of new C. glutamicum-based industrial applications as well as to the refining of its existing capabilities, the current study selected two promoters on the basis of the DNA microarray and following qRT-PCR analyses, which were evaluated and applied successfully for lignocellulolytic enzyme secretion in C. glutamicum.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Strains, plasmids and culture conditions

Strains and plasmids used are listed in Table S1. Ptac, Plac, PmalE1 and Pgit1 were PCR amplified using the primer sets listed in Table S2 and cloned into the SmaI site of pCRB200 (Nishimura et al. 2008), resulting in plasmids pCRD200, 201, 202 and 203, respectively. Xylanase-secretion plasmids, pCRD204 and pCRD205, were constructed as follows: Secretion signal of cgR_2069 (Watanabe et al. 2009) and the GH11 region of the Clostridium cellulovorans xynA gene were PCR amplified using their respective primer sets (Table S2) and ligated at the SnaBI site to produce the chimeric gene, ‘2069xynA’. 2069xynA was then fused downstream of PmalE1 or Pgit1 by crossover PCR using the respective primer sets (Table S2). The resulting PmalE12069xynA and Pgit12069xynA fusion fragments were ligated to the pCRD304 fragment (amplified with primer set 304_PacI_r/304_SpeI_f) at the PacI and SpeI sites, producing pCRD204 and pCRD205, respectively.

Escherichia coli was grown at 37°C in Luria-Bertani (LB) medium (Sambrook et al. 1989). C. glutamicum was precultured in 10 ml rich medium (A medium with 4% glucose), resuspended in 100 ml BT medium containing 2% of glucose, fructose, ribose, gluconate, maltose or sucrose to a final OD610 of 0·15 and cultivated at 33°C (Inui et al. 2007). Where appropriate, E. coli media was supplemented with 50 μg ml−1 of chloramphenicol or ampicillin and C. glutamicum with 5 μg ml−1 chloramphenicol or 50 μg ml−1 kanamycin.

Nucleic acid extraction

DNA and RNA were extracted as described elsewhere (Toyoda et al. 2008). RNA was extracted at mid-log phase (OD610 of 1·0) unless otherwise stated.

DNA microarray

DNA microarray procedures were as described elsewhere (Inui et al. 2007). Mean fluorescence intensity for each spot was an average of four hybridization signals obtained from duplicate spots on two different slides prepared with two independent RNA samples with different combinations of Cy dyes. Genes were considered up- or down-regulated if their signal intensity changed at least twofold. Differences were considered significant if the P-value of a t-test was <0·01.

Quantitative RT-PCR

Quantitative RT-PCR procedures were as described elsewhere (Toyoda et al. 2008).

Each 20 μl reaction contained 10 μl Power SYBR Green Master Mix, 150 nmol l−1 forward/reverse primer (Table S2), 5 U MuLV reverse transcriptase, 8 U RNase inhibitor and 100 ng total RNA (1 ng total RNA for the reference 16S rRNA gene).

Rapid amplification of cDNA ends

SMART RACE cDNA amplification kit (Clontech, San Diego, CA) was used with primers listed in Table S1. RACE products were TA cloned using a pGEM-T Easy Vector System (Promega, Madison, WI) and sequenced using an ABI PRISM 3130xl/3730xl genetic analyser (Applied Biosystems, Foster City, CA).

β-galactosidase assay

The β-galactosidase assay protocol is described elsewhere (Toyoda et al. 2008). For plate assay, C. glutamicum and E. coli harbouring promoter probe vectors were grown on BT (+2% carbon source; +0·02% X-gal) or LB (±1% glucose; +0·02% X-gal; +100 μg ml−1 IPTG (isopropyl-β-d-thiogalactopyranside)) agar plates, respectively. C. glutamicum and E. coli plates were incubated at 33°C for 24 h and 37°C for 48 h, respectively.

RBB-xylan assay

Plate assay: C. glutamicum harbouring pCRD204, pCRD205 and pCRD304 were grown in rich medium until early-stationary phase and resuspended in BT medium to a final OD610 of 0·5. Ten microlitres of each culture was spot inoculated onto RBB-xylan agar plates (BT medium containing 0·2% RBB-xylan (Sigma) and 2% carbon source).

Xylanase activity of culture supernatant: Overnight rich medium cultures of C. glutamicum harbouring pCRD204, pCRD205 and pCRD304 were inoculated in 10 ml BT medium with 2% carbon source to a final OD610 of 0·2. After 24-h cultivation at 33°C, 10 ml of culture supernatant was concentrated to 500 μl using Centricon YM-3 (Millpore, Bedford, MA). The mixture (100 μl each) of substrate solution (10 mg ml−1 RBB-xylan in 100 mmol l−1 Na-acetate buffer; pH 6·0) and sample supernatant (100 μg total protein) was incubated at 37°C for 6 h. Unhydrolysed xylan was precipitated by adding 800 μl of 95% ethanol. The reaction supernatant was collected, and released dye was quantified spectrophotometrically (A570).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Promoter screening by DNA microarray and qRT-PCR

The gene expression profiles of the 3080 predicted C. glutamicum R open reading frames (ORFs) during growth on the disaccharide maltose and the organic acid gluconate were each compared to that of a standard monosaccharide glucose-grown culture. The maltose culture yielded a total of 57 genes that were up-regulated at least twofold. Of these, six were up-regulated at least fourfold (Table 1). The gluconate culture gave a total of 39 genes up-regulated at least twofold, of which three were up-regulated at least fourfold (Table 1). To select the strongest inducible promoters, the gene showing the greatest signal intensity with maltose (cgR_2367, 9·3 × 105) and gluconate (cgR_2459, 2·9 × 105) were chosen for further analysis.

Table 1.   List of Corynebacterium glutamicum genes highly up-regulated (>fourfold) by maltose and gluconate, ordered by signal intensity
CDS*Gene productSignal intensity†Ratio‡
  1. * Coding sequence.

  2. †The values are the averages of four total Cy fluorescence intensities for each spot.

  3. ‡Ratios are calculated relative to the glucose culture.

Up-regulated by maltose
cgR_2367Maltose-binding protein9·3 × 1054·6
cgR_2695Zn-dependent alcohol dehydrogenase3·0 × 10514·1
cgR_0366Putative regulatory protein (WhiB-related protein)1·6 × 1054·2
cgR_2366ABC-type sugar transport system, permease component1·3 × 1055·3
cgR_1469Universal stress protein UspA or related nucleotide-binding protein7·0 × 1044·2
cgR_1897Hypothetical protein3·1 × 1044·5
Up-regulated by gluconate
cgR_2459Galactosides–pentoses–hexuronides (GPH) family sugar transporter2·9 × 10510·7
cgR_2851Putative membrane protein6·2 × 1045·5
cgR_0788ABC-type cobalamin/Fe3+-siderophores transport system, permease component8·6 × 1044·6

Transcripts of cgR_2367 and cgR_2459 were analysed by qRT-PCR to confirm carbon source inducibility observed by DNA microarray. The approximately sixfold increase in the cgR_2367-transcription on maltose relative to glucose at mid-log phase (Fig. 1a) was of the same order as that observed with DNA microarray. The expression levels of cgR_2459 on gluconate on the other hand were significantly higher than that observed with DNA microarray, relative to glucose (Fig. 1b), which could be because of the DNA microarray signals being nearly saturated for higher expressions.

image

Figure 1.  Relative mRNA levels of cgR_2367 (malE1) (a) and cgR_2459 (git1) (b) at mid-log (OD610 of 1·0) and late-log (OD610 of 2·0) phases, relative to the control glucose culture at mid-log phase.

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Molecular analyses of cgR_2367 and cgR_2459

5′-RACE analysis revealed the major transcriptional start site of cgR_2367 and cgR_2459 to be localized 161 and 64 bp, respectively, upstream of the initiation of translation codon (Fig. 2). Putative −35 and −10 regions of cgR_2367 and cgR_2459 and their similarities with the C. glutamicum and E. coliσ70 promoter consensus sequences (Hawley and McClure 1983; Patek et al. 2003b) are indicated.

image

Figure 2.  The promoter region of cgR_2367 (malE1) (a) and cgR_2459 (git1) (b). Transcriptional start points are indicated by a bent arrow marked +1. Putative −10 and −35 elements are boxed with asterisks indicating bases shared either with the Corynebacterium glutamicum consensuses (shown above the boxes; small and capital letters occur more than 40 and 70% of the C. glutamicum consensuses, respectively, and y stands for c/t) or with the Escherichia coliσ70 consensuses (shown underneath the boxes). The putative RBS are underlined. The start codons of malE1 and git1 are marked +161 and +64, respectively. Amino acid sequences are presented below the corresponding coding sequences and boxed. The E. coli maltose box consensus is dot underlined, and a putative cAMP receptor protein-binding motif is double underlined. The numbers 2605558 and 2598790 indicate the position on chromosome.

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cgR_2367 is the first gene of a putative operon containing three other genes (cgR_2363-2365) encoding proteins which are likely involved in sugar transport (Fig. 2a). We named the gene malE1. CgR2367, CgR2365 and CgR2364 showed homology to known binding protein-dependent maltose transport systems such as AmyEDC of Thermoanaerobacterium thermosulfurigenes (Sahm et al. 1996), with 28, 44 and 40% identities, respectively, and MalEFG of E. coli (Duplay et al. 1984), with 22, 27 and 27% identities, respectively. CgR2363 showed no sequence similarity with other known proteins. Upstream of the putative operon is located cgR_2369, whose product showed 47% similarity to MalK of E. coli. A putative maltose box consensus (Bedouelle et al. 1982), 5′-GGAGGG-3′, and a putative binding motif of the cAMP receptor protein (CRP) (Letek et al. 2006), 5′-TGTGG-N6-TCACT-3′, were found in the malE1 promoter region.

CgR2459 showed 25–30% identity to known galactosides–pentoses–hexuronides (GPH) family sugar transporters (Bassias and Bruckner 1998; Chaillou et al. 1998; Liang et al. 2005). We therefore named the cgR_2459 gene git1 (putative gluconate-induced transporter). Unlike these GPH family sugar-transporter genes, however, git1 unlikely forms an operon structure, as no other sugar utilization genes are found in its immediate vicinity. Furthermore, although the presence of a CRE (catabolite responsive element)-like element and mediating CcpA (carbon catabolite control protein A)-dependent catabolite repression is common among known GPH family sugar-transporter proteins (Bassias and Bruckner 1998; Chaillou et al. 1998), no such sequence was detected in the promoter region of git1 (Fig. 2b). Search for a CRP consensus, as was found in the malE1 promoter region, also returned no homology.

Activity of PmalE1 and Pgit1 in Corynebacterium glutamicum and Escherichia coli

Activity of PmalE1 and Pgit1 were evaluated using the promoter probe vector containing a promoterless β-galactosidase reporter gene. PmalE1 and Pgit1 were induced by maltose and gluconate, giving as much as 3·4- and 4·2-fold higher β-galactosidase activities, respectively, relative to glucose (Table 2). Addition of 0·02 or 0·2% glucose repressed activity of PmalE1 and Pgit1 (data not shown). Higher β-galactosidase activity by PmalE1 was also induced by ribose (2·5-fold) and gluconate (2·1-fold). Fructose and sucrose showed similar repressing activity on PmalE1 as glucose. In the case of Pgit1, maltose showed almost the same degree of β-galactosidase activity as gluconate. Slightly higher (1·5-fold) Pgit1 activity was observed with ribose culture, whereas fructose and sucrose showed an even more repressing effect on the promoter, compared to glucose. When grown on glucose, β-galactosidase activity for Ptac (pCRD201) was 7·9 ± 0·9 (miller unit; mean ± SD) and that for Plac (pCRD200) was below the detection limit. PmalE1 and Pgit1 were, therefore, stronger than Ptac by about 30–100-fold and 7–50-fold, respectively, depending on the carbon source used.

Table 2.   Effect of a variety of carbon sources on the malE1 and git1 promoter strength
Carbon source (2%)β-galactosidase activities for*
PmalE1Pgit1
Miller unitsRatio†Miller unitsRatio†
  1. *The values are mean ± SD of three independent experiments.

  2. †Ratios are calculated relative to the glucose culture for each promoter.

Glucose242 (±47·8)1·0 (±0·19)110 (±4·4)1·0 (±0·04)
Maltose826 (±99·5)3·4 (±0·4)460 (±0·7)4·1 (±0·01)
Gluconate528 (±19·1)2·1 (±0·07)468 (±16·2)4·2 (±0·14)
Ribose609 (±58·8)2·5 (±0·24)175 (±4·1)1·5 (±0·03)
Fructose261 (±25·3)1·0 (±0·1) 59 (±14·4)0·5 (±0·13)
Sucrose246 (±15·8)1·0 (±0·06) 55 (±6·5)0·5 (±0·05)

On BT agar plates (+X-gal), C. glutamicum colonies changed from light to dark blue with increasing strength of PmalE1 and Pgit1 activities in response to different carbon sources (Fig. 3a). Plac and Ptac induced no visible β-galactosidase activity in C. glutamicum colonies grown on any of the carbon sources tested, again showing that activities of Plac and Ptac are much lower than those of PmalE1 and Pgit1 especially in the presence of inducing carbon sources.

image

Figure 3.  Effect of a variety of carbon sources on the cgR_2367 (malE1) and cgR_2459 (git1) promoter strength in Corynebacterium glutamicum (a) and heterologous functionality of PmalE1 and Pgit1 in Escherichia coli (b). The plates were inoculated with C. glutamicum (a) and E. coli (b) transformed with pCRD200 (Plac; control), pCRD201 (Ptac; control), pCRD202 (PmalE1) and pCRD203 (Pgit1).

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As expected, the control promoters Plac and Ptac were functional in E. coli, forming dark blue colonies, with the exception of pale blue colonies formed with catabolite-repressed Plac in the presence of glucose (Fig. 3b). β-galactosidase activity was not detected with PmalE1 with or without glucose whereas Pgit1 did function in E. coli but was catabolite repressed by glucose, as was Plac (Fig. 3b).

Use of PmalE1 and Pgit1 for xylanase secretion in Corynebacterium glutamicum

Under the control of PmalE1 and Pgit1, xylanase was successfully produced and secreted by C. glutamicum in response to varying promoter strength caused by carbon sources. On RBB-xylan plates, much larger clearing zones around the colonies were observed with inducible carbon sources (Fig. 4). Xylanase activity of the pCRD204-transformant was 5·3-fold greater with maltose (A570 of 0·23 and 0·043 with maltose and glucose, respectively: Fig. 4a). Likewise, pCRD205-transformant xylanase activity was 3·1-fold greater with gluconate (A570 of 0·038 and 0·12 with glucose and gluconate, respectively: Fig. 4b).

image

Figure 4.  Secretion of xylanase using the cgR_2367 (malE1) and cgR_2459P (git1) promoters. Corynebacterium glutamicum transformants with pCRD204 (PmalE1), pCRD205 (Pgit1) and pCRD304 (negative control) were tested for xylanase secretion.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The DNA microarray gene expression profiling revealed that the ‘carbohydrate transport and metabolism’ category of Clusters of Orthologous Groups contained the most genes up-regulated at least twofold by maltose (9%) and gluconate (15%), indicating that changing the carbon source for C. glutamicum has profound effects on the expression of carbohydrate transport and metabolism genes.

Based on DNA microarray and qRT-PCR analyses, the promoters of cgR_2367 and cgR_2459 were the strongest induced by maltose and gluconate, respectively. In agreement with earlier observations (Patek et al. 2003b), both their −35 regions appear to be only weakly conserved, whereas their −10 consensus appears more responsible for promoter recognition. The respective gene products showed homology to known maltose-binding protein and GPH family sugar transporter. Moreover, the maltose-induced up-regulation of cgR_2367 and its consequent suppression by glucose agree with previous observations for the maltose-binding protein in other bacteria (Sahm et al. 1996; van Wezel et al. 1997). In the E. coli maltose transport system, the malB region of the chromosome encodes two divergent operons (malE-malF-malG and malK-lamB-malM) which are positively controlled by the synergistic action of MalT [binding motif: 5′-GGA(G/T)GA-3′] and CRP (Bedouelle et al. 1982). Gram-positive T. thermosulfurigenes on the other hand exhibits a putative maltose box (AAGGAG) in addition to a CRE motif-like consensus sequence (Sahm et al. 1996). Although the structure of the C. glutamicum maltose-binding region described here revealed only one operon, a few CRP family regulatory proteins are actually annotated on the C. glutamicum R genome, suggesting that the maltose-regulation system of Gram-positive C. glutamicum may be more similar to that of E. coli than to the one of T. thermosulfurigenes. Given that LamB is located in the outer membrane (Benz 1988) and that MalM is a periplasmic protein (Gilson et al. 1986), the absence of a LamB and MalM homologues in C. glutamicum R is consistent with the lack of an outer membrane in Gram-positive bacteria.

The reason why the expression of the git1 gene was induced by gluconate, which does not belong to GPH, is not immediately clear. The gene might be involved in a sugar transport system which works differently from the known systems, so the specific function of the Git1 protein needs to be clarified.

Unlike C. glutamicum promoters reported so far (Patek et al. 2003a,b), the fact that heterologous promoter function of PmalE1 is absent (or very low) in E. coli, and that Pgit1 is repressible by glucose in E. coli is a great advantage when the target gene product has a deleterious effect on E. coli cells transformed with E. coli–C. glutamicum shuttle vectors. We often encounter difficulties in cloning heterologous secretory enzyme genes, such as lignocellulolytic enzyme genes, in E. coli using E. coli–C. glutamicum shuttle vectors, supposedly because of deleterious effect of the secretory enzymes on E. coli membranes. In fact, cloning of xynA from Cl. cellulovorans in E. coli–C. glutamicum shuttle vector under control of Ptac is so far unsuccessful. A similar problem was reported in cloning the DNA fragment encoding secretory protein (miniCipC1) using Cl. acetobutylicum–E. coli shuttle vector (Perret et al. 2004). Owing to functional absence/repressibility of PmalE1/Pgit1 in E. coli, however, xynA GH11 region could be cloned and secreted in C. glutamicum. Activity of secreted xylanase was successfully demonstrated to be induced by maltose and gluconate, using PmalE1 and Pgit1, respectively. Lignocellulolytic enzymes have been gaining increasing importance in the development of biofuel technology, and production of such enzymes in addition to other commercially useful chemicals in industrially useful C. glutamicum as a host could become relevant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to Dr Roy H. Doi (UC Davis) for providing Cl. cellulovorans chromosomal DNA. We thank Crispinus A. Omumasaba for the critical reading of the manuscript. This work was supported by a grant from NEDO, Japan.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Table S1. Strains and plasmids used in this study.

Table S2. Primers used in this study.

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LAM_2776_sm_TableS1-S2.doc96KSupporting info item

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