Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum
© Zhang et al. 2015
Received: 3 March 2015
Accepted: 6 May 2015
Published: 17 May 2015
The shikimic acid (SA) pathway is a fundamental route to synthesize aromatic building blocks for cell growth and metabolic processes, as well as for fermentative production of various aromatic compounds. Genes encoding enzymes of SA pathway are not continuous on genome and they are differently regulated.
In this study, efforts were made to construct continuous genetic modules of SA pathway that are regulated by a same Ptac promoter. Firstly, aro genes [aroG (NCgl2098), aroB (NCgl1559), aroD (NCgl0408) and aroE (NCgl1567)] from Corynebacterium glutamicum and ribosome binding site (RBS) libraries that were tailored for the above genes were obtained, and the strength of each RBS in the 4 libraries was quantified. Secondly, 9 genetic modules were built up from the RBS libraries, a previously characterized ribozyme insulator (RiboJ) and transcriptional promoter (Ptac) and terminator, and aroG, aroB, aroD and aroE. The functionality and efficiency of the constructed genetic modules were evaluated in C. glutamicum by determination of SA synthesis. Results showed that C. glutamicum RES167ΔaroK carrying a genetic module produced 4.3 g/L of SA, which was 54 folds higher compared to that of strain RES167ΔaroK (80 mg/L, without the genetic module) during fermentation in 250-mL flasks. The same strain produced 7.4, and 11.3 g/L of SA during 5-L batch and fed-batch fermentations, respectively, which corresponding to SA molar yields of 0.39 and 0.24 per mole sucrose consumption.
These results demonstrated that the constructed SA pathway modules are effective in increasing SA synthesis in C. glutamicum, and they might be useful for fermentative production of aromatic compounds derived from SA pathway.
KeywordsShikimic acid pathway Corynebacterium glutamicum Shikimate production Synthetic biology Genetic modules Ribosome binding site (RBS)
The development of synthetic biology brings new concepts to design and construct genetic modules or metabolic engineering for bioprocesses. Genetic elements that regulate transcription, translation or encode various enzymes are used as “parts” to build genetic modules [10, 11]. Ideally, the properties of the parts and modules can be accurately and quantitatively predicted when they are implanted into chassis cells [12, 13]. Recently, scientists have designed and constructed a series of parts libraries of promoters, ribosome binding sites (RBS) and terminators, which enabled the regulation of gene expression over wide dynamic ranges in Escherichia coli cells [14, 15]. For example, RBS of different strengths have been applied to optimize the metabolic flux of mevalonate-based farnesyl pyrophosphate biosynthetic pathway . So far, synthetic parts and modules are very limited for C. glutamicum, an important industry production workhorse that has been used for decades to produce amino acids, vitamins, nucleotides [17–20], and recently biofuels and chemicals [21–24].
In this study, efforts were made to construct continuous genetic modules for SA pathway with synthetic biology logistics. Four RBS libraries that were tailored for C. glutamicum and 9 genetic modules for SA synthesis were constructed. The functionality and efficiency of the constructed SA pathway modules were evaluated by determination of SA production with C. glutamicum. Results suggested that the newly constructed pathway modules were effective. During batch and fed-batch fermentation, SA production reached titers of 7.4 and 11.3 g/L, respectively. This represented the highest titer of fermentative production of SA with C. glutamicum.
Design, construction, and screening of RBS libraries for aroB, aroD, aroE and aroG
RBS sequences such as AGAAAGGAGG and GAAAGGAGG [25–27] had been previously identified in C. glutamicum. In addition, the sequence of AAAGGAGGA had been used for expression of genes involving in biopolyester synthesis with C. glutamicum . All these RBS sequences shared a common feature of AAAGGAGG, which is correspondent to the anti-Shine-Dalgarno sequence at the 3’-end of the 16S rRNA from corneybacteria . In addition, it was reported that the spaces between RBS and translational start codon were found to be dominantly 5–10 nucleotides in C. glutamicum . Based on these observations, we generalized a seeding sequence of AAAGG(N)6–9. According to this design, a pool of RBS sequences was chemically synthesized.
Construction and evaluation of genetic modules for SA pathway
Genetic modules of the above 9 combinations were constructed and were inserted into pXMJ19. Thus, 9 pXMJ19 derivatives, namely plasmid-1 to plasmid-9, were obtained and were transferred into C. glutamicum RES167ΔaroK cells. To determine that if gene translations in the genetic modules were exactly correlated to their RBS strengths as they were previously determined, shikimate dehydrogenase (AroE) activities were determined. As shown in (Fig. 4b), those modules (GHBLDLEL, GMBHDMEL, and GLBMDHEL) harbored low strengths of RBS exhibited low AroE activities and those modules (GHBHDHEH, GMBMDLEH, and GLBLDMEH) harbored higher strengths of RBS exhibited higher AroE activities. These results suggested that levels of gene translations in the 9 genetic modules were highly correlated to RBS strengths determined previously via EGFP fluorescence intensities.
Genetic modules increased SA synthesis with C. glutamicum
Bacterial strains and plasmids used in this study
E. coli DH5α
F− endA1thi-1 recA1 relA1 gyrA96deoRΦ80dlacΔ(lacZ) M15 Δ(lacZYA-argF)U169hsdR17(rK −, mK +) λ− supE44 phoA
C. glutamicum RES167
Restriction-deficient mutant of ATCC 13032, Δ(cglIM-cglIR-cglIIR)
University of Bielefeld
Res167 derivate, a fragment of DNA encoding for aroK was deleted
Res167∆aroK derivate, containing plasmid pZB-aroG
Res167∆aroK derivate, containing plasmid pZB-aroB
Res167∆aroK derivate, containing plasmid pZB-aroD
Res167∆aroK derivate, containing plasmid pZB-aroE
Mobilizable vector, for gene disruption in C. glutamicum
University of Bielefeld
Derived from pK18mobsacB, carrying aroK gene
Derived from pK18mobsacB-aroK, a 573 bp fragment of aroK was deleted
pUC19 carrying RiboJ
Plasmid carrying enhanced green fluorescence protein (GFP) gene
Shuttle vector (Camr, Ptac, lacIq, pBL1 oriV C.glu. pK18 oriV E. coli. )
University of Bielefeld
pXMJ19 carrying RiboJ gene
Derived from pXMJ19, carrying both RiboJ and GFP genes
Derived from pZB, carrying aroG MU gene with various RBS
Derived from pZB, carrying aroD MU gene with various RBS
Derived from pZB, carrying aroB MU gene with various RBS
Derived from pZB, carrying aroE MU gene with various RBS
pXMJ19 carrying aroG of which recognition sites of HindIII and PstI were mutated
pXMJ19 carrying aroB of which recognition sites of BamHI and SpeI were mutated
pXMJ19 carrying aroD of which recognition site of PstI were mutated
pXMJ19 carrying aroE of which the recognition sites of EcoRI and SalI were mutated
pXMJ19 carrying RiboJ and aroG MU gene with high strength RBS
pXMJ19 carrying RiboJ and aroG MU gene with medium strength RBS
pXMJ19 carrying RiboJ and aroG MU gene with low strength RBS
pXMJ19 carrying RiboJ and aroB MU gene with high strength RBS
pXMJ19 carrying RiboJ and aroB MU gene with medium strength RBS
pXMJ19 carrying RiboJ and aroB MU gene with low strength RBS
pXMJ19 carrying RiboJ and aroD MU gene with high strength RBS
pXMJ19 carrying RiboJ and aroD MU gene with medium strength RBS
pXMJ19 carrying RiboJ and aroD MU gene with low strength RBS
pXMJ19 carrying RiboJ and aroE MU gene with high strength RBS
pXMJ19 carrying RiboJ and aroE MU gene with medium strength RBS
pXMJ19 carrying RiboJ and aroE MU gene with low strength RBS
Plasmid pXMJ19-RiboJ-aroGMU-H derivate, containing aroB-H module (Ptac-RiboJ-aroB, aroB gene with high strength RBS)
pXMJ19-GHBH derivate, containing aroD-H module (Ptac-RiboJ-aroD, aroD gene with high strength RBS)
pXMJ19-GHBHDH derivate, containing aroE-H module (Ptac-RiboJ-aroE, aroE gene with high strength RBS)
Plasmid pXMJ19-RiboJ-aroG MU-H derivate, containing aroB-M module, aroD-M module, aroE-M module
Plasmid pXMJ19-RiboJ-aroG MU-H derivate, containing aroB-L module, aroD-L module, aroE-L module
Plasmid pXMJ19-RiboJ-aroGMU-M derivate, containing aroB-H module, aroD-M module, aroE-L module
Plasmid pXMJ19-RiboJ-aroG MU-M derivate, containing aroB-M module, aroD-L module, aroE-H module
Plasmid pXMJ19-RiboJ-aroG MU-M derivate, containing aroB-L module, aroD-H module, aroE-M module
Plasmid pXMJ19-RiboJ-aroG MU-L derivate, containing aroB-H module, aroD-L module, aroE-M module
Plasmid pXMJ19-RiboJ-aroG MU-L derivate, containing aroB-M module, aroD-H module, aroE-L module
Plasmid pXMJ19-RiboJ-aroG MU-L derivate, containing aroB-L module, aroD-M module, aroE-H module
Plasmid 2 derivate, containing a terminator between aroB and aroD Module
Plasmid pXMJ19-GBTDE derivate, containing a terminator between aroD and aroE module
Plasmid pXMJ19-GBTDTE derivate, containing a terminator between aroG and aroB module
Insertion of transcriptional terminators into genetic modules further increased SA production with C. glutamicum
SA production in 250-mL flasks and 5-L fermenters with C. glutamicum RES167ΔaroK/pXMJ19-GBTDE
Several methods, such as overexpression of aro genes [31, 32] and the use of enzymes with improved properties , have been reported to enhance the metabolic flux into SA pathway, thus finally increase the production of aromatic amino acids or shikimic acid. This current study revealed a new synthetic biology strategy: Four aro genes were organized as continuous genetic modules and their transcriptions were coordinated by the same tac promoter, RiboJ and terminator. The translation levels of aro genes in the genetic modules were regulated by their RBS, which were quantatively characterized in this study.
RBS is vital to initiate genetic translation, and are useful synthetic biology parts for construction modules . In this study, four tailored-made RBS libraries were constructed and the strength of each RBS sequence was determined in the background of C. glutamicum cells. Although the RBS libraries were tailored for aroG, aroB, aroD and aroE, it is believed that these RBS would be applicable also for other purposes when C. glutamicum was used as host. Similarly, the constructed SA pathway modules were tested for SA production in this study, they should be also useful for productions such as aromatic amino acids that are derived from SA pathway.
SA is a highly valued commercial compound. Efforts were made to improve SA production by de-repressing of feedback inhibition of enzymes involved in SA synthesis , increasing glucose availability , and optimizing metabolic fluxes , with E. coli or B. subtilis. So far as we know, C. glutamicum has not been exploited for SA production. By implementing the constructed genetic modules in the shikimate kinase deficient mutant, C. glutamicum was successfully engineered to produce SA at 11.3 g/L in 5-L fermenter. So far, this represents the highest titer of SA production with C. glutamicum. The SA production with C. glutamicum is comparable to the productivity with B. subtilis (19.7 g/L) . Although this SA titer is lower when compared to SA production by E. coli (84 g/L) , C. glutamicum is still a promising SA producer due to its non-pathogenic nature, and its productivity can be further improved by optimization of fermentation process, or by replacement of the tryptophan- and prephenate-sensitive DAHP synthase [36, 37].
Synthetic biology tool boxes for manipulating C. glutamicum were expanded by including 4 RBS libraries, in addition to the previous reported promoters [38, 39] and CoryneBrick . The RBS libraries represent the first set of RBS libraries that were quantatively characterized in C. glutamicum. The selected RBS and aro genes could be organized as continuous genetic modules and their transcriptions could be coordinated. Genetic modules were successful constructed for SA pathway, and were demonstrated to be useful for increase of SA synthesis. In fed-batch fermentation, C. glutamicum harboring newly constructed SA pathway modules achieved 11.3 g/L SA, which represented the highest SA production with C. glutamicum.
Materials and methods
Microorganisms, plasmids, medium, and cultivation
The bacterial strains and plasmids used in this study are listed in Table 1. C. glutamicum was cultivated at 30 °C in Luria Bertani (LB)  broth or Brain Heart Infusion (BHI) medium . E. coli was cultivated at 37 °C in 50 mL of LB broth in 250-ml flasks on a rotary shaker at 200 rpm. When needed, chloramphenicol at a final concentration of 10 or 20 μg/mL in medium was used for cultivation of C. glutamicum or E. coli. Expression of genes with C. glutamicum was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).
Fermentative production of shikimic acid with C. glutamicum was carried out in 250 mL flasks and 5-L fermenter (Bioflo Model 3000 bioreactor, New Brunswick Scientific, N.J., U.S.A.). Seeding cultures were grown with Medium A (g/L): K2HPO4 · 3H2O (0.5); KH2PO4 (0.5); (NH4)2SO4 (10); glucose (40); MgSO4 · 7H2O (0.2); phenylalanine (0.15); tyrosine (0.15); tryptophan (0.15); CaCO3 (30); FeSO4 · 7H2O (0.02); MnSO4 · 4H2O (0.02); biotin (50 μg); thiamine (200 μg), pH 7.4.
Fermentation was conducted with Medium B (g/L): K2HPO4 · 3H2O (0.5); KH2PO4 (0.5); Urea (3); sucrose (38); MgSO4 · 7H2O (0.2); Yeast extract (10); peptone (4); FeSO4 · 7H2O (0.02); MnSO4 · 4H2O (0.02); biotin (50 μg); thiamine (200 μg), pH 7.4. The fermenter was stirred at 300 rpm, aerated at 3.0 vol/vol per minute, and pH was maintained at 7.0. Cell growth was monitored by measuring optical density at 600 nm (OD600) with a spectrophotometer (Biospec-1601 DNA/Protein Enzyme Analyzer, Shimadzu). Cellular dry weights were determined by centrifugation and lyophilization with 3 parallel samples.
C. glutamicum was cultivated in mineral salts (MS) medium when RBS strength were tested. The MS medium contained following components (g/L, pH 8.0): Na2HPO4 · 12H2O (2); KH2PO4 (0.5); MgSO4 · 7H2O (0.03); NH4C1 (0.53); trace element solution 2 mL. Trace element solution (g/L, pH 6.0): EDTA, (0.5); ZnSO4 · 7H2O, (0.22); CaCl2, (0.055); MnCl2 · 4H2O, (0.051); FeSO4 · 7H2O, (0.0499); (NH4)6Mo7O24 · 4H2O, (0.011); CuSO4 · 5H2O, (0.0157); CoCl2 · 6H2O, (0.0161); biotin (0.0125); thiamine (0.05).
DNA extraction, amplification, plasmid construction and genetic transformation
Plasmid and chromosomal DNAs were isolated using the OMEGA Plasmid Mini Kit and the OMEGA Bacterial DNA Kit (Omega genetics, Beijing), respectively. DNA fragments from PCR amplification were purified with the OMEGA Cycle-Pure Kit (Omega genetics, Beijing). Restriction enzymes, ligases and other DNA-manipulating enzymes were used according to their manufacturer’s instructions. Genetic transformation of C. glutamicum and E. coli was carried out by electroporation, and recombinant strains were selected according to Tauch et al. .
Construction of pXMJ19-aroG MU, pXMJ19-aroD MU pXMJ19-aroB MU, pXMJ19-aroE MU and pZB
Oligonucleotides used in this study
Amplification of aroG from genome, SalI and KpnI underlined
Amplification of aroB from genome, SalI and KpnI underlined
Amplification of aroD from genome, SalI and KpnI underlined
Amplification of aroE from genome, PstI and KpnI underlined
Mutate HindIII in aroG
Mutate PstI in aroG
Mutate PstI in aroG
Mutate BamHI in aroB
Mutate SpeI in aroB
Mutate PstI in aroD
Mutate EcoRI in aroE
Mutate SalI in aroE
Amplification of RiboJ from pUC19, HindIII and PstI underlined
Amplification of egfp from pACGFP, KpnI and EcoRI underlined
Amplification of aroG with mutated RBS, SalI and KpnI underlined
Amplification of aroB with mutated RBS, SalI and KpnI underlined
Amplification of aroD with mutated RBS, SalI and KpnI underlined
Amplification of aroE with mutated RBS, SalI and KpnI underlined
aroG with high strength RBS, SalI and BamHI underlined
aroG with medium strength RBS, SalI and BamHI underlined
aroG with lows trength RBS, SalI and BamHI underlined
aroB with high strength RBS, SalI and BamHI underlined
aroB with medium strength RBS, SalI and BamHI underlined
aroB with low strength RBS, SalI and BamHIunderlined
aroD with high strength RBS, SalI and BamHI underlined
aroD with medium strength RBS, SalI and BamHI underlined
aroD with low strength RBS, SalI and BamHI underlined
aroE with high strength RBS, SalI and BamHI underlined
aroE with medium strength RBS, SalI and BamHI underlined
aroE with low strength RBS, SalI and BamHI underlined
BamHI and XmaI underlined
XmaI and KpnI underlined
KpnI and EcoRI underlined
EcoRI and HindIII underlined
Primer used to verify ΔaroK
pZB was derived from pXMJ19. Chemically synthesized gene of RiboJ (27) was cloned into pXMJ19 at HindIII and PstI sites, resulting in pXMJ19-RiboJ. This pXMJ19-RiboJ was digested with EcoRI and KpnI, and a genetic fragment encoding the enhanced green fluorescence protein was cloned at the KpnI and EcoRI sites. The resulting plasmid was named pZB, and was used for later construction of RBS libraries.
Design and construction of RBS libraries tailored for aroG, aroB, aroD and aroE, and evaluation of RBS strength according to fluorescence intensity
Based on the currently known RBS sequences from C. glutamicum, we designed a seeding sequence of AAAGG(N)6–9, where “N” represents any nucleotide of A, T, G, or C. From this seeding sequence, oligonucleotides tagged as MU-RBSAG-F, MU-RBSAB-F, MU-RBSAD-F, and MU-RBSAE-F, were chemically synthesized. These oligonucleotides and their partner primers (Table 2) were used to amplify the aro genes from plasmid pXMJ19-aroG MU, pXMJ19-aroB MU, pXMJ19-aroD MU, pXMJ19-aroE MU. The amplified aro genes, each had a specific RBS sequence at its 5’-end, were digested with restriction endonuclease and were cloned into the samely digested pZB. Thus, four RBS libraries were constructed and were named as pZB-aroG, pZB-aroB, pZB-aroD, and pZB-aroE (Fig. 2).
The strength of each RBS for genetic translation was determined according to its fluorescence intensity. C. glutamicum cells harboring single plasmid (thus a single RBS) of libraries of pZB-aroG, pZB-aroB, pZB-aroD, and pZB-aroE were cultivated in the presence of 0.5 mM IPTG at 30 °C in MS medium. After incubation for 48 h at 30 °C and 200 rpm, 200 μl of cell suspension was transferred into a 96-well plate. The fluorescence from the eGFP in C. glutamicum cells and optical density were measured using a BioTek® synergy H4 Hybrid Reader (Keruiente, Beijing, China).
Construction of genetic modules for SA pathway
To construct the nine plasmids with the combination of different strength RBS, aroG gene with high, middle and low strength RBS were amplified from pXMJ19-aroG MU and cloned between SalI and BamHI cloning sites of plasmid pXMJ19-RiboJ. These three plasmids were named as pXMJ19-RiboJ-aroG MU-H, pXMJ19-RiboJ-aroG MU-M and pXMJ19-RiboJ-aroG MU-L, respectively. Taking the same way, we got plasmids pXMJ19-RiboJ-aroB MU-H, pXMJ19-RiboJ-aroB MU-M, pXMJ19-RiboJ-aroB MU-L, pXMJ19-RiboJ-aroD MU-H, pXMJ19-RiboJ-aroD MU-M, pXMJ19-RiboJ-aroD MU-L, pXMJ19-RiboJ-aroE MU-H, pXMJ19-RiboJ-aroE MU-M and pXMJ19-RiboJ-aroE MU-L, which also have the high, middle and low strength RBS, accordingly. Then, Ptac-RiboJ-aroB MU-H fragments with BamHI and XmaI sites were amplified from plasmid pXMJ19-RiboJ-aroB MU-H and cloned into plasmid pXMJ19-RiboJ-aroG MU-H, resulting plasmid named pXMJ19-GHBH. Then fragments Ptac-RiboJ-aroD MU-H with XmaI and KpnI sites were cloned into plasmid pXMJ19-GHBH, resulting plasmid named pXMJ19-GHBHDH. From plasmid pXMJ19-RiboJ-aroE MU-H we got fragments Ptac-RiboJ-aroE MU-H with KpnI and EcoRI sites and cloned the fragments into plasmid pXMJ19-GHBHDH, resulting plasmid named plasmid-1. Plasmid-2 to plasmid-9 and derivate plasmids were also got by the way describe above. Three terminator fragments with XmaI, BamHI and KpnI cloning sites were amplified from plasmid pXMJ19, respectively. After terminator with XmaI site was cloned in plasmid-2, we got plasmid pXMJ19-GBTDE. Then terminator with BamHI site was cloned in plasmid pXMJ19-GBTDE to get plasmid pXMJ19-GBTDTE. Plasmid pXMJ19-GTBTDTE was constructed by cloning terminator with KpnI site.
Measurement of SA dehydrogenase activity
The enzyme activities of the shikimate dehydrogenases were assayed by monitoring the absorbance of NADPH at 340 nm (ε = 6230 M−1 cm−1) using a spectrophotometer (Specord 205 Analytik, Jena, Germany). The assays were conducted at 25 °C in a volume of 1 mL solution, containing 100 mM Tris–HCl buffer at pH 8.0, 1 mM SA, and 2 mM NADP+. Cellular lysates from C. glutamicum were added finally to trigger the reaction. One unit of enzyme activity was defined as the amount of enzyme catalyzing the conversion of 1 μmol of NADP+ per minute at 25 °C.
For preparation of cellular lysates of C. glutamicum, cells were harvested by centrifugation (6000 g, 4 °C, 5 min) of culture samples. Supernatants were removed, the cell pellets were washed and re-suspended in 50 mM pH 8.0 Tris–HCl buffer. This cell suspension was subjected to sonication (Ningbo Scientz Biotechnology Co., LTD, China) and centrifugation (12,000 g, 4 °C, 10 min). The supernatants were collected and used for enzyme assays. Protein concentrations were determined using Bradford method .
Construction of C. glutamicum RES167∆aroK
Disruption of the shikimate kinase gene, aroK, in C. glutamicum was performed using the suicide vector pK18mobsacB. The intact DNA fragment (2946 bp) of aroK was amplified from chromosomal DNA of C. glutamicum, using the primers aroK-F and aroK-R (Table 1). This intact aroK fragment was cloned into pK18mobsacB EcoRI/HindIII sites. The resulting plasmid was named pK18mobsacB-aroK, and was amplified with primers KTaroK-F and KTaroK-R, thus resulting DNA fragments with disrupted aroK gene. After digested with XmaI restriction endonuclease, DNA fragments were ligated and transformed into E. coli. The recombinant plasmid was named pK18mobsacB-∆aroK and was electroporated into C. glutamicum RES167. Using the method described by Schäfer et al. , the aroK mutant RES167∆aroK was screened out on BHI agar plates. The Disruption of aroK was verified by PCR amplification and sequence of the disrupted aroK gene from RES167∆aroK.
Determination of SA and 3-dehydroshikimic acid concentrations
The concentrations of SA and 3-dehydroskimic acid were determined with an HPLC system (Agilent 1200 series, Agilent Technologies, Inc., USA) equipped with a ZORBAX SB C18 column (4.6 mm x 250 mm x 5 μm) and detected at 215 nm wavelength. The HPLC was run with a mixture of solution A (phosphoric acid in water, pH 2.5) and solution B (methanol) as eluant and was operated at a flow rate of 0.35 mL/min. The following gradient was used: at 0–7.5 min, 95 % of solution A and 5 % of solution B; at 7.5-15 min, 100 % of solution B; 15.0-22.5 min, 95 % of solution A and 5 % of solution B. Standard shikimic acid (Cat. No. S5375, Sigma-Aldrich, USA) and 3-dehydroshikimic acid (Cat. No. 05616, Sigma-Aldrich, USA) were eluted at 5.411 and 6.241 min, respectively, under these conditions.
Determination of sucrose concentrations
The sucrose concentrations in fermentation broth were determined with spectrometric method, as previously described .
This work was supported by 973 Project from Ministry of Science and Technology (No. 2012CB7211-04).
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