Generation of aroE overexpression mutant of Bacillus megaterium for the production of shikimic acid
© Ghosh and Banerjee; licensee BioMed Central. 2015
Received: 9 December 2014
Accepted: 6 May 2015
Published: 17 May 2015
Shikimic acid, the sole chemical building block for the antiviral drug oseltamivir (Tamiflu®), is one of the potent pharmaceutical intermediates with three chiral centers. Here we report a metabolically engineered recombinant Bacillus megaterium strain with aroE (shikimate dehydrogenase) overexpression for the production of shikimic acid.
In a 7 L bioreactor, 4.2 g/L shikimic acid was obtained using the recombinant strain over 0.53 g/L with the wild type. The enhancement of total shikimate dehydrogenase activity was 2.13-fold higher than the wild type. Maximum yield of shikimic acid (12.54 g/L) was obtained with fructose as carbon source. It was isolated from the fermentation broth using amberlite IRA-400 resin and 89 % purity of the product was achieved.
This will add up a new organism in the armory for the fermentation based production which is better over plant based extraction and chemical synthesis of shikimic acid.
Shikimic acid, with three chiral centers is regarded as a versatile hydroaromatic intermediate for the pharmaceutical industry. Since the synthesis of Oseltamivir (TamifluR) from shikimic acid, its industrial demand has increased exponentially . Due to its unique structure, it has been used as the building block for the synthesis of several biologically active compounds such as antibiotics, antitumor agents , antithrombotic agents [3, 4], and vitamins etc. It has also been used in the organic synthesis and cosmetic industry . Currently, shikimate is mainly produced by chemical synthesis or extraction from the fruit of Illicium spp. However, these processes are complicated with the high cost and/or limitations of raw materials making it difficult to meet the increasing worldwide requirements due to the global pandemic of influenza . Microbial fermentation is regarded as a potential alternative for large scale production considering the increasing demand of shikimate .
In this study, we have developed a modified strain of Bacillus megaterium with aroE (shikimate dehydrogenase) overexpression for the production of shikimic acid (Fig. 1). The aroE gene is targeted for this study, as shikimate dehydrogenase is one of the rate limiting steps in the shikimic acid pathway. Various carbon sources were used to study their effect on the yield of shikimic acid. As no computational model was available for shikimate dehydrogenase of B. subtilis, a homology model was developed. To rationalize the model, docking study was performed using dehydroshikimate as substrate.
Results and discussion
Expression profile of shikimate dehydrogenase and PMF based identification
Generation of homology model and docking study
Docking studies were carried out with dehydroshikimate as ligand to show the binding interaction of substrate with the enzyme. The pocket finder analysis showed that Lys 111, Met 263, Lys 264 play the crucial role in substrate binding and activity (Fig. 3c).
Shikimate dehydrogenase activity with aroE overexpression strain
Shikimic acid production using recombinant strain
To evaluate the effect of aroE overexpression on the growth and shikimic acid production, a batch reactor of 7 L capacity was run in LB medium with the recombinant B. megaterium. The cells were induced with 2 % xylose, when the OD600nm reached to 0.4 and cell mass was 0.8 g/L. During the growth and production of shikimic acid by the recombinant organism, (Fig. 4) reducing sugar concentration started decreasing from the very beginning and it was found to be 1.6 g/L at 24 h. Xylose was used for induction and it seems that the most of the xylose had been utilized by B. megaterium for the growth and shikimic acid production. The maximum shikimic acid concentration was 4.2 g/L at the end of 24 h as analyzed and quantified by HPLC. DO concentration showed the downward trend initially along with the increase of growth and finally it reached to the saturation level at the end of fermentation. The initial pH of the fermentation medium was 6.5 and during course of growth and shikimic acid production, it was found that the pH of the fermentation broth was on the increasing side. The cell mass concentration started increasing from the very beginning and a maximum of 6.6 g/L cell mass was obtained at 24 h.
Comparison of fermentation parameters of the wild type and recombinant strain in the growth and production of shikimic acid
Qv (g SA/L.h)
Qp (mg SA/g cell mass.h)
Y(X/S) (g/100 g)
Y(P/S) (g/100 g)
Effect of carbon sources
Shikimic acid yields with respect to cell mass under different carbon sources
Shikimic acid (g/L)
Isolation of shikimic acid and its identification
Anion exchange resin, amberlite IRA-400 Cl− was used for the extraction of shikimic acid from the fermentation broth following the method as described in methods section. After extraction, the product was characterized as shikimic acid by HPLC, Mass and NMR (Additional file 1: Supporting information: S1, S2, S3, S8, S9, S10).
The effect of overexpression of aroE gene (shikimate dehydrogenase) on the yield of shikimic acid is reported in this paper. The maximum shikimic acid yield of 12.54 g/L was achieved using the recombinant strain at 7 L reactor level. The effect of various carbon sources was investigated and fructose was found to be the most effective carbon source for shikimic acid accumulation. The homology model developed for shikimate dehydrogenase has been used for the docking study and the enzyme-substrate interaction is shown. The amino acid residues involved in substrate binding and catalysis were identified. The study shows that over expression of aroE gene has significant effect over the shikimic acid yield in B. megaterium than the previously reported strains. By this single alteration in the pathway, an improved Bacillus strain is developed which is better over some of the recombinant counterparts with multiple modifications. Though the yield is lower than the reported values, with further modifications of the recombinant strain (such as generation of aroK knock out), this may be developed as a potential process for industrial application. There is a possibility of having higher titer of shikimic acid on the achievement of complete optimization studies with the recombinant B. megaterium.
Culture and growth condition
B. megaterium MTCC 428 and B. subtilis MTCC 441 was obtained from Microbial Type Culture Centre, Institute of Microbial Technology, Chandigarh, India. E. coli Top10, available in the laboratory, was used as cloning host. Luria Bertani broth (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) was used for the growth of both the organisms. Various concentrations of antibiotics (ampicillin 100 μg/mL, tetracycline 10 μg/mL) was used in the medium for the selection of recombinants of E. coli and B. megaterium, respectively. Cells were grown in an incubator shaker (Kuhner, Germany) at 37 °C (200 rpm) for 12 h (E. coli) and 24 h (B. megaterium).
➢ Fermentation media and growth condition
For the production of shikimic acid using wild type and recombinant strain, in shake flask and bioreactor, Luria Bertani broth was used. Induction was carried out with 2 % xylose (w/v) at OD600nm of 0.4 (cellmass 0.8 g/L). Tetracycline (10 μg/mL) was used for the selective growth of the recombinant. For shake flask experiments, the strain (glycerol stock stored at −80 °C) was grown in 20 mL LB-Tet medium as starter culture. This fresh culture was used to inoculate (2 %, v/v) 100 mL medium at 37 °C (200 rpm). For 7 L bioreactor (5 L working volume), 250 mL starter culture was prepared for inoculation. Glucose feeding of 50 g/L was given after 24 h, in the stationary phase. The reactor was aerated at 0.5 VVM and the dissolved oxygen concentration was maintained above 30 % air saturation by agitation at 200–500 rpm. Antifoam (polypropylene glycol) was added manually as and when needed.
aroE-FP: 5′ GATGCACTAGTATGAAAAAGCTGTACGGGGTTATCGG 3′
aroE-RP: 5′ GATGCGGTACCTTAACATTCTGTTCCTCCTAATTTTCC 3′
Transformation of vector construct into protoplast of B. megaterium
The transformation of B. megaterium protoplasts provides an elegant method to introduce foreign plasmid DNA . For the transformation of protoplasts, method reported by Biedendieck et al.  was followed. After transformation, the protoplasts were platted on Tet-LB-agar plate and incubated at 37 °C. Colonies were observed indicating the successful transformation.
Overexpression of shikimate dehydrogenase (aroE)
In plasmid pWH1520, the promoter is under the control of xylose induction. For the expression of cloned gene, xylose concentration was optimized. Different concentrations of xylose (1, 2, 3, 4, 6 %, w/v) were used. Cells were induced at OD600nm of 0.2-0.4. Cells were harvested after 24 h, once stable OD600nm was achieved. Cells were washed twice with phosphate buffer (100 mM, pH 7.0) and resuspended in lysis buffer (100 mM Tris–HCl, 0.4 mM DTT, 1.2 mM PMSF, 1 mM EDTA) at final cellmass concentration of 100 mg/mL. Cells were disrupted by French Press at 600 psi and the suspension was centrifuged to remove the cell debris. Both the pellet and supernatant was used as sample for PAGE (12 %) to check for the protein.
Identification of over expressed protein
The over expressed protein was characterized by peptide mass fingerprinting using in-gel digestion method, as reported by Shevchenko et al. . The peptide profile was checked by MALDI analysis and compared with the MASCOT database . The MASCOT score was used for the identification of protein.
Homology modelling of shikimate dehydrogenase and docking study
Homology model was developed on the basis of sequencing data of the cloned aroE gene using the Expasy server and Swiss Dock model . The model was validated by Prochek. Docking was carried out with Swiss Dock system using dehydroshikimate (from Zinc database) as ligand. Binding site of the ligand was predicted using pocket finder and docking analysis. Binding interaction of the ligand with the enzyme was predicted using Chimera map.
Assay for shikimate dehydrogenase (SDH)
The enzyme was assayed in the reverse direction using shikimic acid as substrate at 25 °C by monitoring the reduction of NAD+ at 340 nm . The assay mixture (total volume 1 mL) contained 100 mM Na2CO3 (pH 10.6), 4 mM shikimic acid and 2 mM NAD+. Substrate blank, enzyme blank and co-factor blank was used for each assay experiment. One unit of enzyme activity is defined as the amount of enzyme that catalyses the conversion of 1 μmol of substrate/min.
Cell growth was monitored by measuring the optical density at 600 nm. Glucose concentration was estimated by DNS method . Shikimic acid concentration was determined by HPLC using Waters Alliance e2695 series instrument and Alltech OA-2000 organic acid column (100 × 6.5 mm, 6.5 μm) (Grace Davison Discovery Science, Deerfield, Illinois, USA) maintained at 30 °C. The mobile phase was 0.005 N H2SO4 with a flow rate of 0.5 mL/min. Shikimic acid was detected at 254 nm with a photodiode detector and quantified using a standard curve.
Isolation of shikimic acid from fermentation broth
Shikimic acid being anionic in nature, an anion exchange chromatography was used for its extraction from the fermentation broth. Amberlite IRA-400 Cl− was used for this purpose . Cells were separated by centrifugation; supernatant was concentrated on rotavapor till a viscous mass was formed, dissolved in methanol and centrifuged again. The resultant solution was then dried in rotavapor till it formed a layer. The solid residue then re-dissolved in water and filtered. The filtrate was loaded onto an Amberlite IRA-400 chloride column and washed with deionized water. Shikimic acid was eluted with 25 % aqueous acetic acid and concentrated on rotavapor. The concentrate was analyzed by HPLC, GC-MS and NMR.
SG gratefully acknowledges the Indian Council of Medical Research, Govt. of India for financial support. SG acknowledges Satyajeet Salunkhe (NIPER, SAS Nagar, India) for helping in the docking study. SG and UCB gratefully acknowledge Mukesh Yadav (NIPER, SAS Nagar, India) for his assistance in running the fermenter.
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