Development and Characterization of Expression Vectors for Corynebacterium glutamicum
Development and Characterization of Expression Vectors for Corynebacterium glutamicum
Journal of Microbiology and Biotechnology. 2014. Jan, 24(1): 70-79
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : October 10, 2013
  • Accepted : October 27, 2013
  • Published : January 28, 2014
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Jinho, Lee

In an attempt to develop a variety of expression vector systems for Corynebacterium glutamicum , six types of promoters, including P tac , P sod , P sod with a conserved Shine-Dalgarno (SD) sequence from C. glutamicum , P ilvC , P ilvC with a conserved SD-1 (P ilvC-M1 ), and P ilvC with a conserved SD-2 (P ilvC-M2 ), were cloned into a modified shuttle vector, pCXM48. According to analysis of promoter strength by quantitative reverse transcription PCR, P sod and P sod-M were superior to tac and ilvC promoters in terms of transcription activity in C. glutamicum . All of the promoters have promoter activities in Escherichia coli , and P sod-M displayed the highest level of transcriptional activity. The protein expression in constructed vectors was evaluated by measuring the fluorescence of green fluorescent protein (GFP) and SDS-PAGE. C. glutamicum harboring plasmids showed GFP fluorescence with an order of activity of P ilvC > P ilvC-M1 > P sod > P ilvC-M2 > P sod-M , whereas all plasmids except pCSP30 with P sod displayed fluorescence activities in E. coli . Of them, the strongest level of GFP was observed in E. coli with P sod-M , and this seems to be due to the introduction of the conserved SD sequence in the translational initiation region. These results demonstrate that the expression vectors work well in both C. glutamicum and E. coli for the expression of target proteins. In addition, the vector systems harboring various promoters with different strengths, conserved SD sequences, and multiple cloning sites will provide a comfortable method for cloning and gene expression, and consequently contribute to the metabolic engineering of C. glutamicum .
Corynebacterium glutamicum is a gram-positive soil bacterium that has been widely used in the industrial production of many amino acids, including monosodium glutamate and lysine [10] . Recently, with the accumulation of rapidly increasing information and techniques regarding C. glutamicum , such as genetic manipulation tools [12 , 22 , 34 , 35] , whole genome information [11 , 15] , functional genomic techniques [38 , 40] , and integration of systems biology into metabolic engineering [3 , 18] , C. glutamicum is becoming regarded as one plausible microorganism for the large production of bio-based chemicals, materials, and fuels including D-ornithine, 2-ketoisovalerate, succinate, cadaverine, putrescine, 1,2-propanediol, ethanol, 1-butanol, and polygalacturonic acid [1] . Additionally, since C. glutamicum belongs to the GRAS (generally regarded as safe) microorganisms, it can be applicable to production of foodor pharmaceutical-grade proteins [7] .
Bacterial promoters play a crucial role in the expression and regulation of genes regarding production of valuable metabolites or proteins in microorganisms [26 , 41] . The well-known Escherichia coli promoters such as tac , trc , lac UV5, and P R , P L promoters have been used for gene expression in C. glutamicum [5 , 24 , 29 , 36] . The expression by these promoters was inducible following the addition of lactose and its analog IPTG, arabinose [16] , or acetic acid [6] . Although the E. coli promoters were active in C. glutamicum , they display very weak activities and the transcriptional regulation of gene expression was relatively inefficient when compared with E. coli [29] . For the purpose of efficient modulation of gene expression, many endogenous promoters from C. glutamicum have been isolated and characterized based on the promoter sequences in the -30 and -10 regions and regulation mechanisms [17 , 22 , 26 , 39] . The promoters of sod gene coding for superoxide dismutase [2 , 23 , 27] , eftu encoding elongation factor tu [2] , dapA coding for dihydrodipicolinate synthase [39] , and gdh encoding glutamate dehydrogenase [9] were employed for metabolic engineering of C. glutamicum to produce several metabolites. However, to date, suitable expression vector systems with endogenous strong promoters were limited, and most were utilized for replacement of promoters of genes within the chromosome sequences. Besides this, the strength of promoters described above, along with tac promoter at the transcriptional level, was not compared and evaluated with each other in corynebacteria. On one side, many reports illustrate that protein expression levels are strongly influenced by the mRNA secondary structure and the short Shine-Dalgarno (SD) sequence in the 5’-untranslated region (5’-UTR) of bacterial mRNAs as well as the promoter strength [4 , 8 , 14 , 25 , 30 , 33] , and so, it is difficult to choose appropriate promoter(s) for optimal expression of each gene [41] . In this sense, it is necessary to construct several expression vector systems harboring a variety of promoters with different strengths, conserved SD sequence, and multiple cloning sites comfortable for gene cloning and expression.
In this study, I report the construction of expression vector systems for C. glutamicum with three types of promoters, P tac , P sod , and P ilvC , which are known to function in C. glutamicum , and verified its capability through the analyses of quantitative reverse transcription PCR (qRT-PCR) and protein expression using green fluorescent protein (GFPuv). Furthermore, I developed novel expression vector systems in which the conserved SD sequence of C. glutamicum was introduced into promoters P ilvC and P sod and confirmed that these systems function in both C. glutamicum and E. coli .
Materials and Methods
- Bacterial Strains, Plasmids, and Culture Conditions
Bacterial strains and plasmids used in this study are described in Table 1 . E. coli Top 10 was employed as the host for general DNA manipulation. The DNA template of sod and ilvC promoters was obtained from C. glutamicum ATCC 13032, whereas pKK223-3 and pGFPuv were used as DNA templates for obtaining P tac -multiple cloning sites-4 (MCS-4)-T rrnB fragment and gfp gene, respectively. MCS-2 was obtained from an E. coli / C. glutamicum shuttle vector, pCES208 [24] . The E. coli / C. glutamicum shuttle vector pXMJ19 and its derivatives [13 , 22] were used for the construction of expression vector systems with several promoters. E. coli and C. glutamicum were grown in LB medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl) at 37℃ and 32℃, respectively. If necessary, 25 and 4.5 μg/ml of chloramphenicol were added to the culture media of E. coli and C. glutamicum , respectively. In the case of E. coli Top 10/pCTG20, when cell OD reached about 0.4, 1 mM of IPTG was added into the LB medium.
The bacterial strains and plasmids used in this study.
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aATCC, American Type Culture Collection. bAmpR, ampicillin resistance; KanR, kanamycin resistance; CmR, chloramphenicol resistance.
- Recombinant DNA Techniques and Transformation
All the general recombinant DNA techniques were carried out according to Sambrook et al . [31] . Restriction enzymes, pfu -x DNA polymerase, plasmid mini-prep kit, and gel extraction kit were purchased from New England Biolab (USA), Solgent Corp. (Korea), Intron (Korea), and Macrogen (Korea), respectively. Primer sequences used in this study are listed in Table 2 . All PCR constructs were verified by DNA sequencing. Plasmid DNA was transformed into C. glutamicum by electroporation [37] .
Primer sequences used in this study.
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aRestriction enzyme sites are underlined.
- Subcloning of pXMJ19
To remove P tac , rrn B transcriptional terminator (T rrnB ), and the lacI q gene in pXMJ19 and insert MCS-2 of pCES208 into modified pXMJ19 by polymerase chain reaction (PCR), a 4.7 kb fragment from pXMJ19 and 0.12 kb MCS-2 in pCES208 were amplified by using primer sets P1-P2 and P3-P4, respectively ( Fig. 1 A). Both fragments were gel-purified, digested with Not I and Nhe I, and ligated with each other. The resulting plasmid, pCXM48, was introduced into E. coli Top 10, and transformants were selected on chloramphenicol-containing LB agar plates ( Fig. 1 B).
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Schematic representation of recombinant plasmids. (A) Plasmid map of pXMJ19 with Ptac, MCS-1, and TrrnB. The MCS-1 contains the following restriction sites (5’→3’ direction); HindIII, PstI, SalI, XbaI, BamHI, SmaI, KpnI, and EcoRI. (B) Plasmid map of pCXM48 with the MCS-2 site from pCES208. MCS-2 contains the following restriction sites; NotI, XbaI, BamHI, PstI, EcoRI, EcoRV, HindIII, SalI, KpnI, and NheI. (C) Plasmid map of the Ptac-containing expression vector pCXT20. MCS-3 has NotI-XbaI- KpnI-NheI restriction sites; MCS-4 has EcoRI, BamHI, PstI, and HindIII sites. TrrnB means rrnB transcriptional terminator.
- Construction of Expression Vectors
A P tac -T rrnB from pKK223-3 was cloned into pCXM48 by PCR using primers P5 and P6. A 0.59 kb PCR product digested by Xba I and Kpn I was ligated with pCXM48/ Xba I/ Kpn I, and yielded pCXT20 ( Fig. 1 C). To construct P sod - and P ilvC -containing expression vectors, 0.3 kb of P sod and P ilvC fragments were amplified by using primer sets P7-P8 and P9-P10, respectively, and gel-purified. The digested products were then cut with Xba I and Eco RI, cloned into the same restriction sites of pCXT20 in which P tac was removed, generating pCXS30 and pCXI40, respectively ( Fig. 3 )
- Construction of Expression Vectors with Conserved SD Sequence of C. glutamicum
To introduce the conserved SD sequence of C. glutamicum in expression vectors with sod and ilvC promoters, fragments P sod-M , P ilvC-M1 , and P ilvC-M2 from the chromosomal DNA of C. glutamicum were amplified using primer sets P7-P11, P9-P12, and P9-P13, respectively ( Fig. 2 ). The resulting purified fragments cut by Xba I and Eco RI were then cloned into the Xba I/ Eco RI-cleaved pCXT20 in which P tac was removed to produce pCXS35, pCXI43, and pCXI45, respectively ( Fig. 3 ).
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Comparison of DNA sequences in 3’-regions of Ptac, Psod, and PilvC. Underlined italic characters are the putative SD sequence or conserved SD sequence introduced in the 3’-regions of each promoter. The underlined lower case letters are the EcoRI site introduced into the wild-type sequence.
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Schematic diagram of expression vectors. pCXS30, pCXS35, pCXI40, pCXI43, and pCXI45 contain promoters Psod, Psod with a conserved SD; PilvC, PilvC with a conserved SD-1; and PilvC with a conserved SD-2, respectively.
- Construction of GFPuv-Containing Expression Vectors
A gfp gene was cloned using the constructed expression vectors. A 0.7 kb product amplified by using primers P14 and P15 was inserted into Eco RI/ Hin dIII-digested pCXT20, pCXS30, pCXS35, pCXI40, pCXI43, and pCXI45, respectively, and designated pCTP20, pCSP30, pCSP35, pCIP40, pCIP43, and pCIP45.
- Measurement of GFPuv Fluorescence
Overnight cultures using recombinant E. coli and C. glutamicum cells harboring expression vectors with gene gfp in LB medium were harvested by centrifugation at 5,000 ×g for 10 min, washed three times with PBS buffer (NaCl 4 g/l, KCl 0.1 g/l, Na 2 HPO 4 0.72g/l, KH 2 PO 4 0.12 g/l, pH 7.4), and then resuspended in 1 ml of the same buffer. A bead beator (Biospec Product, Inc.) disrupted the cells, and cell debris was removed by centrifugation at 13,000 × g for 30 min, yielding crude extracts, which were used for the measurement of fluorescence. GFPuv fluorescence was measured by using a spectrofluorophotometer (Shimadzu, RF-5300PC) with excitation at 395 nm and emission at 508 nm. All of the measurements were performed using independent cultures three times.
- Analysis of Protein Concentration and SDS-PAGE
Protein concentration in crude extracts was determined by using the Bio-Rad protein assay kit (Bio-Rad, USA) with bovine serum albumin as the standard. Protein expression was monitored using 10% SDS-PAGE. Native SDS-PAGE was performed as follows. Loading samples of native state were prepared by adding a loading dye (0.21 M Tris-HCl, pH 8.45, 11% glycerol, 0.8% SDS, 0.004% Coomassie blue G, 0.004% phenol red) into crude extracts and incubated for 2h at 37℃. After running the SDS-PAGE, the gel was washed three times with 10 mM Tris-HCl buffer (pH 8.0) for 1 h, and then GFP fluorescence was monitored at 260 nm of UV light.
- Quantitative Reverse Transcription PCR (qRT-PCR)
For analysis of transcriptional levels of gfp in expression vectors, cells were cultivated to the mid-exponential growth phase (OD 0.8 ~1.0), and total RNA was extracted from cells using the IQeasy RNA extraction Mini kit (Intronbio, Korea) according to the manufacturer’s instructions and stored at -72℃. Reverse transcription was performed by using SuperScript II (Invitrogen). Following reverse transcription, cDNA was amplified by using primer pairs P16-P17 (GFP primer set), P18-P19 ( C. glutamicum 16S rDNA primer set), and P20-P21 ( E. coli 16S rDNA primer set), respectively. Realtime PCR was performed in a Step One Plus machine (Applied Biosystems (AB)) using the SYBR Green PCR kit (AB), according to the manufacturer’s instructions in a total volume of 20 μl. Cycling conditions for amplification of GFP, C. glutamicum 16S rDNA ( C. glutamicum internal control), and E. coli 16S rDNA ( E. coli internal control) were 10 min at 95℃, 40 cycles of 15 sec at 95℃, and 30 sec at optimal Tm (59℃). Quantification was carried out with StepOne software v.2.2.2 (AB). The relative GFP expression levels were analyzed using the 2 -(ΔΔCT) method [20] , normalized to 16S rDNA expression of C. glutamicum or E. coli and represented as x-fold increase in a recombinant cell harboring pCSP30, pCSP35, pCIP40, pCIP43, or pCIP45 (sample ΔC T ) compared with the corresponding cell with pCTP20 (positive control ΔC T ).
Results and Discussion
- Development of Vector Systems from pBL1 Family
The typical autonomously replicating vectors for C. glutamicum are based on the small cryptic plasmids pBL1 and pCG1 from C. glutamicum [22 , 32] . Both vector systems are compatible in corynebacteria, and so it enables the study of the genetics, physiology, and metabolic engineering of C. glutamicum . Since the widely utilizing restriction enzyme sites including Eco RI and Hin dIII are present in pCG1 family plasmids, it is preferable to use pBL1 family plasmids for the construction of expression vectors [13] . In this work, I constructed expression vector systems based on plasmid pXMJ19 ( Fig. 1 A), a pBL1 family, as follows. First, the arrangement of MCS-1 in pXMJ19 is different to that of pKK223-3, which carries the tac promoter that is widely used for gene expression in E. coli , which led researchers to clone genes inconveniently using both E. coli and C. glutamicum . Thus, I deleted P tac -MCS-1-T rrnB from pXMJ19 and cloned P tac -MCS-4-T rrnB into a newly constructed vector, pCXM48 ( Fig. 1 B). Second, because the constitutive expression systems are superior to the inducible systems in economical aspects, the lacI q gene was deleted from pXMJ19. Third, to conveniently clone genes in both pBL1 and pCG1 families, the MCS-2 of pCES208, which has the same replication origin of pCG1, was introduced into the modified pXMJ19. To do this, I constructed pCXM48 by deleting P tac - MCS-1-T rrnB and lacI q and introducing MCS-2 of pCES208 into the subcloned pXMJ19 ( Fig. 1 B). Finally, an expression vector, pCXT20, was developed by cloning of P tac -MCS-4-T rrnB from pKK223-3 into pCXM48 ( Fig. 1 C).
- Development of Expression Vector Systems
Two promoters, P sod and P ilvC , together with P tac described above were selected based on the following reasons. First, the promoter of the sod gene was extensively used for metabolic engineering of C. glutamicum by exchanging the native promoters of the dld , pyc , malE , dapB , lysC , and tkt genes for the sod promoter, which resulted in a marked increase of L-lysine production [23] . Second, the ilvC promoter had one of the highest CAT (chloramphenicol acetyltransferase) activities isolated from the chromosomal library of C. glutamicum using a promoter-probe vector [26] . To facilitate gene cloning and expression, six bases in the 3’-terminus of a 30 nucleotide (nt) sequence in each promoter were replaced by an Eco RI sequence ( Fig. 2 ). As a result, pCXS30 and pCXI40 with P sod and P ilvC , respectively, were constructed ( Fig. 3 ). Meanwhile, the efficiency of translation initiation is known to be crucial for high-level expression of proteins, and is greatly influenced by the accessibility of ribosome to the SD sequence around the translation-initiation region of bacterial mRNAs [25 , 30] . The putative SD sequences in P sod and P ilvC were presumed to be 5’-GAAAGGATT-3’ and 5’-GAAAGGCGA-3’ ( Fig. 2 ), respectively, whereas the consensus SD sequence in C. glutamicum was proposed to be 5’-GAAAGGAGG-3’ [21] . To enhance the efficiency of translational initiation of protein in the constructed expression vector systems, the putative SD sequence of P sod was replaced with 5’-GAAAGGAGG-3’ to yield pCXS35. In addition, two types of SD sequences, 5’-GAAAGGAGA-3’ and 5’-GAAAGGAGG-3’, were introduced in the presumed SD sequence of P ilvC , resulting in plasmids pCXI43 and pCXI45, respectively ( Fig. 3 ).
- Promoter Strength Analysis by qRT-PCR
To evaluate the promoter strength at the transcriptional level, mRNA transcripts of gene gfp in six plasmids were measured by qRT-PCR in C. glutamicum ( Fig. 4 and Supplementary Table S1). The relative transcript level of GFP by P sod was about 2.7 times higher than those for P tac and P ilvC , which indicates that the sod promoter is superior to ilvC and tac promoters with respect to mRNA biosynthesis at the transcriptional level. Besides this, the mRNA transcript level of P tac was similar to that of P ilvC , which was known to be a strong promoter in C. glutamicum [26] . This result demonstrates that P tac in C. glutamicum functions as a strong promoter with a high transcriptional activity. The P sod and P sod-M displayed the same average expression levels, whereas P ilvC-M1 and P ilvC-M2 exhibited increased levels through mutations of the SD region in P ilvC . The GFP transcript levels for the six expression vectors were also evaluated by qRT-PCR in E. coli ( Fig. 4 and Supplementary Table S1). The average transcript levels by P sod and P ilvC promoters were 1.3 times and 3.6 times lower than that by the strong tac promoter, respectively, which imply that P sod and P ilvC originating from C. glutamicum are functional and can synthesize mRNA transcript in E. coli . Interestingly, the transcript level under the control of P sod-M was 5.9 and 7.6 times higher relative to those under P tac and P sod promoters. Thus, according to analysis of the qRT-PCT, sod and sod -M promoters showed the strongest transcriptional activity in C. glutamicum . In particular, the sod -M promoter had the highest level of promoter activity in both C. glutamicum and E. coli , which may facilitate efficient cloning and characterization of interesting genes/proteins in both strains.
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Relative GFP expression level by quantitative reverse transcription PCR. Relative GFP expression level means 2-(ΔΔCT), which was calculated from the number of cycles required for the fluorescent signal to reach threshold (CT). CT values of 16S rDNAs from C. glutamicum and E. coli were used for normalization among samples. The relative GFP expression levels represent the x-fold increase in a recombinant cell harboring pCSP30, pCSP35, pCIP40, pCIP43, or pCIP45 (sample ΔΔCT) compared with the corresponding cell with pCTP20 (positive control ΔΔCT). All error bars represent the value of standard deviations, which were calculated from three experiments on the same sample in the same PCR reaction.
Indeed, I expected that the promoter strength at the transcription level is not influenced by variations in the ribosome binding sites of each promoter; however, the increases in transcription activity were observed in C. glutamicum with P ilvC-M1 and P ilvC-M2 as well as in E. coli harboring P sod-M . By contrast, C. glutamicum having P sod-M along with E. coli containing P ilvC-M1 and P ilvC-M2 showed similar transcriptional promoter activities compared with the activity of the corresponding wild-type promoter for each strain. It has been suggested that the expression of heterologous proteins in recombinant cells is affected by various factors: gene dosage, promoter strength, mRNA stability, and the efficiency of translation initiation [4 , 14] . It seems that variations of transcriptional activity in mutant promoters result from the change of mRNA stability in mutant promoters to different sensitivity for endonucleases and exonucleases in each strain [14] .
- Expression Analyses by GFP Fluorescence Intensity and SDS-PAGE
To compare the developed expression vector systems at the translational level, GFPuv was expressed in the six vectors and then its expression strengths were analyzed using crude extracts of recombinant C. glutamicum . Cells harboring plasmids with promoters P sod , P sod-M , P ilvC , P ilvC-M1 , and P ilvC-M2 showed higher fluorescence intensities, with an order of activity of P ilvC > P ilvC-M1 > P sod > P ilvC-M2 > P sod-M ( Fig. 5 A). However, variants with more conserved SD sequences of C. glutamicum , including P sod-M , P ilvC-M1 , and P ilvC-M2 , exhibited lower GFP fluorescence intensities than recombinant cells with the wild-type promoter P sod or P ilvC . Meanwhile, C. glutamicum with pCTP20 expressing GFP under the control of P tac did not express GFP. The GFP expression of each clone in C. glutamicum was also confirmed by SDS-PAGE. Denatured crude extracts of C. glutamicum harboring plasmids did not show a distinct band corresponding to the molecular mass of about 27 kDa ( Fig. 6 A). When crude extracts were run on SDS-PAGE and refolded, the fluorescence could be detected in cells harboring pCSP30, pCSP35, pCIP40, pCIP43, and pCIP45 ( Fig. 6 B), and this result demonstrates that GFP is expressed in the developed expression vector systems. The expression levels (RFI/mg-protein) of all recombinant C. glutamicum with pCIP series in the LB medium were maintained consistently with culture time (data not shown), which means that the GFP expression by these promoters was constitutive. The expression strength was also analyzed in E. coli . Cells with P sod-M , P ilvC-M1 , and P ilvC-M2 revealed a large increase of GFP fluorescence over the corresponding strain with wild-type promoter ( Fig. 5 B). In particular, cells bearing pCSP35 with GFP attached to P sod-M displayed a 3.3-fold increase of fluorescence intensity compared with the positive control cells harboring pCTP20 in which GFP was linked to the tac promoter. The introduced SD sequences in pCSP35, pCIP43, and pCIP45 showed a high identity with the consensus SD sequence of E. coli (5’-AGGAGGT-3’) [14 , 19] yielding a strong expression of GFP. A noticeable band with about 27 kDa appeared in the denatured state of crude extract from E. coli Top 10 with pCTP20, pCSP35, or pCIP43 ( Fig. 6 C). In addition, crude extracts from three types of cells displayed a bright fluorescence band on SDS-PAGE after refolding ( Fig. 6 D). These results coincided strongly with the result of GFP fluorescence intensity results. Considering that analysis of GFP expression, the constructed vectors were working in C. glutamicum , and especially P sod-M and P ilvC-M1 mediated a strong expression of GFP in E. coli along with C. glutamicum . To conclude, plasmids pCXS-35 and pCXI43 with sod -M and ilvC -M1 promoter, respectively, provide substantive GFP expressions at both the transcription and translation levels in C. glutamicum and E. coli .
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GFP fluorescence intensities of expression vectors. (A) Relative fluorescence intensity (RFI) of C. glutamicum containing vector expressing GFP. (B) Relative fluorescence intensity of E. coli containing vector expressing GFP. The fluorescence of GFP-harboring crude extracts was measured by using spectrofluorophotometry with excitation at 395 nm and emission at 508 nm. Control means cells harboring pCXM48.
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SDS-PAGE of GFPuv expression in recombinant C. glutamicum and E. coli. (A) SDS-PAGE of GFP expression with denatured crude extracts of C. glutamicum. (B) SDS-PAGE of GFP expression with crude extracts from C. glutamicum. After running on PAGE, proteins were refolded by washing with 10 mM Tris-HCl for 1 h. (C) SDS-PAGE of GFP expression with denatured crude extracts of E. coli. (D) SDS-PAGE of GFP expression with crude extracts from E. coli. After running on PAGE, proteins were refolded by washing with 10 mM Tris-HCl for 1 h. Proteins were separated by 10% SDS. Lanes: M, protein size marker; C, pCXM48; Ptac, pCTP20; Psod, pCSP30; Psod-M, pCSP35; PilvC, pCIP40; PilvC-M1, pCIP43; PilvC-M2, pCIP45. The protein loading amounts on gels in A, B, C, and D were 10, 20, 10, and 10 μg, respectively.
When target proteins were expressed in C. glutamicum for metabolic engineering, proteins were usually expressed and characterized in E. coli and then expressed in C. glutamicum using other expression vectors working in C. glutamicum . It is necessary to provide additional genetic manipulation for fine-tuning protein expression. In this sense, the expression vector systems, including those with sod -M and ilvC -M1 promoters, will afford efficient cloning and expression of interesting proteins in C. glutamicum without additional genetic work for metabolic engineering.
According to results regarding promoter strength analyses by qRT-PCR, GFP fluorescence, and SDS-PAGE, I found that GFP expression at the transcription level was not completely correlated with that at the translation level. Recently, many studies have demonstrated that protein expression levels are strongly influenced by the mRNA secondary structure and the accessibility of ribosome to the SD sequence around the translational-initiation region (TIR), as well as by the promoter strength [25 , 30 , 33] . Romasi and Lee [28] demonstrated that although the tac promoter has a strong transcriptional activity, IpdC was well expressed by P tac but not AspC, whereas the sod promoter mediated the expression of AspC but not IpdC in E. coli . This suggests that the weak expression of AspC by P tac is caused by a more stable mRNA secondary structure of TIR in P tac - aspC than that in P sod - aspC . Hence, the mismatch between transcription activity and protein expression in this study was also caused by various factors such as promoter strength, mRNA stability, and the efficiency of translation initiation.
In conclusion, I developed expression vector systems for C. glutamicum with three types of promoters and their derivatives, which are known to function in C. glutamicum , and characterized each promoter’s capability at the transcriptional and translational levels by analyses of qRTPCR, GFP fluorescence, and SDS-PAGE. All the expression vectors work in both C. glutamicum and E. coli , which would facilitate efficient cloning and characterization of interesting genes/proteins in E. coli , at first, and then implement metabolic engineering of C. glutamicum without additional genetic works for fine-tuning of protein expression. In addition, the developed expression vectors and pCES208, another shuttle vector, have the same multiple cloning sites and different replication origins, which will be able to conveniently clone and express many target genes in C. glutamicum with two different vectors for over-production of valuable metabolites or proteins. I expect that the developed expression vector systems will apply to the study of genetics, physiology, and metabolic engineering of C. glutamicum .
This research was supported by Kyungsung University Research Grants in 2013.
Becker J , Wittmann C 2012 Bio-based production of chemicals, materials and fuels - Corynebacterium glutamicum as versatile cell factory. Curr. Opin. Biotechnol. 23 631 - 640    DOI : 10.1016/j.copbio.2011.11.012
Becker J , Klopprogge C , Zelder O , Heinzle E , Wittmann C 2005 Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl. Environ. Microbiol. 71 8587 - 8596    DOI : 10.1128/AEM.71.12.8587-8596.2005
Becker J , Zelder O , Häfner S , Schröder H , Wittmann C 2011 From zero to hero-design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production. Metab. Eng. 13 159 - 168    DOI : 10.1016/j.ymben.2011.01.003
Berg L , Lale R , Bakke I , Burroughs N , Valla S 2009 The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5’-untranslated part of mRNA. Microb. Biotechnol. 2 379 - 389    DOI : 10.1111/j.1751-7915.2009.00107.x
Billman-Jacobe H , Wang L , Kortt A , Steward D , Radford A 1995 Expression and secretion of heterologous proteases by Corynebacterium glutamicum. Appl. Environ. Microbiol. 61 1610 - 1613
Cortay JC , Nègre D , Galinier A , Duclos B , Perrière G , Cozzone AJ 1991 Regulation of the acetate operon in Escherichia coli: purification and functional characterization of the IclR repressor. EMBO J. 10 675 - 679
Date M , Itaya H , Matsui H , Kikuchi Y 2006 Secretion of human epidermal growth factor by Corynebacterium glutamicum. Lett. Appl. Microbiol. 42 66 - 70    DOI : 10.1111/j.1472-765X.2005.01802.x
de Smit MH , van Duin J 1994 Control of translation by mRNA secondary structure in Escherichia coli. A quantitative analysis of literature data. J. Mol. Biol. 244 144 - 150    DOI : 10.1006/jmbi.1994.1714
Hänssler E , Müller T , Palumbo K , Patek M , Brocker M , Krämer R , Burkovski A 2009 A game with many players: control of gdh transcription in Corynebacterium glutamicum. J. Biotechnol. 142 114 - 122    DOI : 10.1016/j.jbiotec.2009.04.007
Hermann T 2003 Industrial production of amino acids by coryneform bacteria. J. Biotechnol. 104 155 - 172    DOI : 10.1016/S0168-1656(03)00149-4
Ikeda M , Nakagawa S 2003 The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl. Microbiol. Biotechnol. 62 99 - 109    DOI : 10.1007/s00253-003-1328-1
Jäger W , Schäfer A , Pühler A , Labes G , Wohlleben W 1992 Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans. J. Bacteriol. 174 5462 - 5465
Jakoby M , Ngouoto-Nkili CE , Burkovski A 1999 Construction and application of new Corynebacterium glutamicum vectors. Biotechnol. Tech. 13 437 - 441    DOI : 10.1023/A:1008968419217
Jana S , Deb JK 2005 Strategies for efficient production of heterologous proteins in Escherichia coli. Appl. Microbiol. Biotechnol. 67 289 - 298    DOI : 10.1007/s00253-004-1814-0
Kalinowski J , Bathe B , Bartels D , Bischoff N , Bott M , Burkovski A 2003 The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. J. Biotechnol. 104 5 - 25    DOI : 10.1016/S0168-1656(03)00154-8
Khlebnikov A , Risa O , Skaug T , Carrier TA , Keasling JD 2000 Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture. J. Bacteriol. 182 7029 - 7034    DOI : 10.1128/JB.182.24.7029-7034.2000
Kim HJ , Kim TH , Kim Y , Lee HS 2004 Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum. J. Bacteriol. 186 3453 - 3460    DOI : 10.1128/JB.186.11.3453-3460.2004
Kohlstedt M , Becker J , Wittmann C 2010 Metabolic fluxes and beyond - systems biology understanding and engineering of microbial metabolism. Appl. Microbiol. Biotechnol. 88 1065 - 1075    DOI : 10.1007/s00253-010-2854-2
Komarova AV , Tchufistova LS , Dreyfus M , Boni IV 2005 AU-rich sequences within 5’ untranslated leaders enhance translation and stabilize mRNA in Escherichia coli. J. Bacteriol. 187 1344 - 1349    DOI : 10.1128/JB.187.4.1344-1349.2005
Livak KJ , Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2-(ΔΔCT)method. Methods 25 402 - 408    DOI : 10.1006/meth.2001.1262
Martín JF , Barreiro C , González-Lavado E , Barriuso M 2003 Ribosomal RNA and ribosomal proteins in corynebacteria. J. Biotechnol. 104 41 - 53    DOI : 10.1016/S0168-1656(03)00160-3
Nešvera J , Pátek M 2011 Tools for genetic manipulations in Corynebacterium glutamicum and their applications. Appl. Microbiol. Biotechnol. 90 1641 - 1654    DOI : 10.1007/s00253-011-3272-9
Neuner A , Heinzle E 2011 Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering. Biotechnol. J. 6 318 - 329    DOI : 10.1002/biot.201000307
Park JU , Jo JH , Kim YJ , Chung SS , Lee JH , Lee HH 2008 Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes. J. Microbiol. Biotechnol. 18 639 - 647
Park YS , Seo SW , Hwang S , Chu HS , Ahn JH , Kim TW 2007 Design of 5’-untranslated region variants for tunable expression in Escherichia coli. Biochem. Biophys. Res. Commun. 356 136 - 141    DOI : 10.1016/j.bbrc.2007.02.127
Pátek M , Eikmanns BJ , Pátek J , Sahm H 1996 Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif. Microbiology 142 1297 - 1309    DOI : 10.1099/13500872-142-5-1297
Ravasi P , Peiru S , Gramajo H , Menzella HG 2012 Design and testing of a synthetic biology framework for genetic engineering of Corynebacterium glutamicum. Microb. Cell Fact. 11 147 - 157    DOI : 10.1186/1475-2859-11-147
Romasi EF , Lee J 2013 Development of indole-3-acetic acidproducing Escherichia coli by functional expression of IpdC, AspC, and Iad1. J. Microbiol. Biotechnol. 23 1726 - 1736    DOI : 10.4014/jmb.1308.08082
Salim K , Haedens V , Content J , Leblon G , Huygen K 1997 Heterologous expression of the Mycobacterium tuberculosis gene encoding antigen 85A in Corynebacterium glutamicum. Appl. Environ. Microbiol. 63 4392 - 4400
Salis HM , Mirsky EA , Voigt CA 2009 Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27 946 - 950    DOI : 10.1038/nbt.1568
Sambrook J , Russell DW 2001 Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY
Santamaría R , Gil JA , Mesas JM , Martín JF 1984 Characterization of an endogenous plasmid and development of cloning vectors and a transformation system in Brevibacterium lactofermentum. J. Gen. Microbiol. 130 2237 - 2246
Seo SW , Yang J , Jung GY 2009 Quantitative correlation between mRNA secondary structure around the region downstream of the initiation codon and translational efficiency in Escherichia coli. Biotechnol. Bioeng. 104 611 - 616    DOI : 10.1002/bit.22431
Suzuki N , Inui M , Yukawa H 2007 Site-directed integration system using a combination of mutant lox sites for Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 77 871 - 878    DOI : 10.1007/s00253-007-1215-2
Tauch A , Pühler A , Kalinowski J , Thierbach G 2003 Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics. J. Biotechnol. 104 27 - 40    DOI : 10.1016/S0168-1656(03)00157-3
Tsuchiya M , Morinaga Y 1988 Genetic control systems of Escherichia coli can confer inducible expression of cloned genes in coryneform bacteria. Nat. Biotechnol. 6 428 - 430    DOI : 10.1038/nbt0488-428
van der Rest ME , Lange C , Molenaar D 1999 A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl. Microbiol. Biotechnol. 52 541 - 545    DOI : 10.1007/s002530051557
Vasco-Cárdenas MF , Baños S , Ramos A , Martín JF , Barreiro C 2013 Proteome response of Corynebacterium glutamicum to high concentration of industrially relevant C4 and C5 dicarboxylic acids. J. Proteomics 85 65 - 88    DOI : 10.1016/j.jprot.2013.04.019
Vasicová P , Pátek M , Nesvera J , Sahm H , Eikmanns B 1999 Analysis of the Corynebacterium glutamicum dapA promoter. J. Bacteriol. 181 6188 - 6191
Wendisch VF 2003 Genome-wide expression analysis in Corynebacterium glutamicum using DNA microarrays. J. Biotechnol. 104 273 - 285    DOI : 10.1016/S0168-1656(03)00147-0
Yim SS , An SJ , Kang M , Lee J , Jeong KJ 2013 Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum. Biotechnol. Bioeng. 110 2959 - 2969    DOI : 10.1002/bit.24954