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Site-directed mutagenesis and over expression of aroG gene of Escherichia coli K-12
International Journal of Biological Macromolecules 51 (2012) 915–919
Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Site-directed mutagenesis and over expression of aroG gene of Escherichia coli K-12 Songyi Lin a,b,1 , Xiujuan Meng a,1 , Jie Jiang a,1 , Daxing Pang b , Gregory Jones c , Hongsheng OuYang b,∗ , Linzhu Ren b,∗∗ a
Laboratory of Nutrition and Functional Food, College of Quartermaster Technology, Jilin University, Changchun 130062, PR China Post-doctoral Research Station of Biology, Jilin University, Changchun 130062, PR China c Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC 29634, USA b
a r t i c l e
i n f o
Article history: Received 25 June 2012 Accepted 11 July 2012 Available online 20 July 2012 Keywords: aroG Site-directed mutagenesis Over-expression
a b s t r a c t 3-Deoxy-d-arabino-heptulonate-7-phosphate (DAHP) synthetase is one of the key enzymes, which catalyzes the first step in the aromatic amino acid biosynthetic pathway and yields the three amino acids tyrosine, tryptophan, and phenylalanine. In Escherichia coli (E. coli), three differently regulated DAHP synthases carry out the first regulated step in the aromatic amino acid biosynthetic pathway. The three DAHP synthases encoded by the genes aroG, aroF and aroH are inhibited by phenylalanine, tyrosine and tryptophan, respectively. In this work, the aroG gene was cloned and mutated by site-directed mutagenesis using splicing overlap extension PCR (SOE-PCR) technique. The feedback-resistant DAHP synthase encoded by aroG was achieved by replacing the residue Leu175 of aroG with Asp as to increase net carbon flow down the common pathway. SDS-PAGE which was used to access the protein expression level of aroGM showed the strain harbored mutated aroGM gene achieve over-expression compared to strain contain empty plasmid pET-28b (+). © 2012 Elsevier B.V. All rights reserved.
1. Introduction The aromatic amino acids, l-tryptophan, l-phenylalanine, and l-tyrosine, can be manufactured by bacterial fermentation. lTryptophan is an essential amino acid that is required for the biosynthesis of proteins and is the precursor for several important biological compounds [1,2]. l-Tryptophan is also an important
Abbreviations: PEP, phosphoenolpyruvate; E4P, erythrose 4-phosphate; DAHP, 3-deoxy-d-arabino-heptulosonate-7-phosphate; PCR, polymerase chain reaction; SOE-PCR, splicing overlap extension polymerase chain reaction; LB, Luria Bertani; IPTG, isopropyl--d-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; AP, ammonium persulfate; TEMED, N,N,N ,N tetramethylethylenediamine; PMSF, phenylmethanesulfonyl fluoride. ∗ Corresponding author. Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi’an Road, Changchun 130062, PR China. Tel.: +86 131 59680552; fax: +86 431 86758018. ∗∗ Corresponding author. Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi’an Road, Changchun 130062, PR China. Tel.: +86 431 87836175; fax: +86 431 86758018. E-mail addresses: ouyanghongsheng [email protected] (H. OuYang), [email protected] (L. Ren). 1 These authors contributed equally to this study and should be regarded as first joint author. 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.07.013
amino acid in terms of its applications in the pharmaceutical, food, and cosmetics industries [3,4]. l-Tryptophan serves as a medicine to improve sleep state, mood and is a food additive. In addition, other industrially relevant substances can be derived from tryptophan synthesis intermediates, like indigo [5] or the anti-influenza drug Oseltamivir phosphate [6]. Chemical synthesis, enzymatic/microbial conversion and microbial fermentation are the methods used for industrial production of l-tryptophan. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to strain improvement. The first application of recombinant DNA technology to strain improvement for l-tryptophan production was performed with Escherichia coli by Tribe and Pittard [7]. They reported increased production by amplification of the trp operon with a deregulated trpE gene, though the final titer of about 1 g/L was low. On the other hand, Aiba et al. constructed a genetically engineered strain by introducing a plasmid containing the trp operon with deregulated trpE and trpD genes into an E. coli trpR and tnaA mutant [8]. The feedback-resistant anthranilate (ANTA) synthase encoded by trpED was achieved by replacing the residue Ser40 of TrpE with Phe [9,10]. The biosynthesis of l-tryptophan is initiated by the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP). 3Deoxy-d-arabino-heptulonate-7-phosphate synthetase (DAHPS) is
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one of the key enzymes, which catalyzes the first step in the aromatic amino acid biosynthetic pathway and yields the three amino acids tyrosine, tryptophan, and phenylalanine. In E. coli, there are three DAHPS isoforms, each specifically inhibited by one of the three aromatic amino acids [11,12]. DAHPS (Phe), a homotetramer encoded by the aroG gene [13], is the major isoform, constituting about 80% of the total activity of the cell [14–16]. The less abundant isozymes, DAHPS (Trp) (1% of total activity) and DAHPS (Tyr) (20% of total activity) are homodimers encoded by the aroH and aroF genes, respectively [17]. 3-Deoxy-d-arabino-heptulosonate-7-phosphate is catalyzed to generate chorismate through three reactions, and then the l-tryptophan branch pathway leading to l-tryptophan. The feedback inhibition site of aroG gene consists of Asp6, Asp7, Ile10, Ilel3, Prol50, Gin151, Leul75, Leul79, Ser180, Phe209, Ser211 and Val221. The replacement of residue Leu175 of aroG with Asp generated phenylalanine-insensitive form of AroG and showed a higher DAHPS activity in the presence of 1 mM phenylalanine compared to the wide-type DAHPS [18]. Based on the previous study, we attempted to introduce defined genetic manipulations into E. coli to develop an l-Trp-producing strain based on known regulatory and metabolic information. The feedback-resistant DAHP synthase encoded by aroG was achieved by replacing the residue Leu175 of aroG with Asp as to increase net carbon flow down the chorismate and more importantly is to increase l-tryptophan production. The feedback-resistant aroG genes was named aroGM and was assembled on the vectors pET-28b (+) and realized over-expressed in E. coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used in the study to test the expression of protein. 2. Materials and methods 2.1. Bacterial strains and plasmids The genome of wild-type E. coli K-12 was used as template to obtain the aroG using polymerase chain reaction (PCR) technology. The pET-28b (+) vectors which were modified as expression vector carry an N-terminal His·Tag/thrombin/T7·Tag configuration plus an optional C-terminal His·Tag sequence. Bacterial strains and plasmids used in this study are listed in Table 1. Strains and relevant details of their properties is also described. 2.2. Media and manipulations The Ex Taq DNA polymerase, T4 DNA ligase and restriction endonucleases were purchased from MBI Fermentas (Burlington, ON, Canada). The pGM-T vector was purchased from TIANGEN (Beijing, China). All enzymatic reactions were performed according to Table 1 Bacterial strains and plasmids used in this study. Strains/plasmids E. coli Escherichia coli k-12 E. coli BL21 (DE3) E. coli Top10
Plasmid pGM-T pET-28b (+) pET-28b (+)-aroGM
Relevant characteristics
Source Lab. save
−
−
ompT, hsdSB (rB mB ), gal dcm(DE3) mcrA (mrr-hsdRMS-mcrBC) 80 lacZM15lacX74 recA1 ara 139 (ara-leu) 7697galU galK rpsL (Strr) endA1 nupG
TIANGEN
Ampr Kanr Contains the aroGM gene
TIANGEN Lab. save This work
TIANGEN
the manufacturers’ instructions. E. coli cells were transformed by heat shock using frozen competent cells prepared using the calcium chloride method described in Molecular Cloning [19]. Plasmid DNA was extracted from E. coli using the Biopin plasmid DNA Extraction Kit (BioFlux, China). Primer Premier 5.0 was used to design primer and DNAMAN was used to achieve sequence alignment. The E. coli strains its derivatives were grown aerobically on a shaker at 37 ◦ C for 12 h in Luria Bertani (LB) medium which contained 10 g/L trypeptone; 10 g/L NaCl; 5 g/L yeast extract or on LB plates which contained10 g/L trypeptone; 10 g/L NaCl; 5 g/L yeast extract and 15 g/L agar powder. When required, 100 g ampicillin/mL or kanamycin was added. 2.3. Cloning and expression of aroGM 2.3.1. Nucleotide sequence accession numbers The complete genome sequences of E. coli str. K-12 substr. MG1655 studied in this paper are available from NCBI under accession number U00096, the sequences of the aroG gene are deposited in NCBI under version numbers GI: 48994873. 2.3.2. The cloning of aroGM gene The aroGM12 (1–525 bp) and aroGM34 (525–1053 bp) genes of E. coli MG1655 were amplified by splicing overlap extension polymerase chain reaction (SOE-PCR) in Thermal Cycler (Thermo Electron Corporation, Milford, MA, USA) with the thermal program of 1 cycle of 94 ◦ C for 5 min, 30 cycles of 94 ◦ C for 1 min, 55 ◦ C for 30 s, 72 ◦ C for 50 s, and 1 cycle of 72 ◦ C for 10 min. Fifty microliter of each reaction mixture contained 1 L each forward and reverse primers (aroGM12 with 5 -CCATGGTAGGATGCTCCTGTTATGGTCGTTATGTCGG-3 and 5 -AGCCCTGATGCATCTTCGCGGTGCAC-3 , and aroGM34 with 5 -GTGCACCGCGAAGATGCATCAGGGCT-3 and 5 GGATCCTTACCCGCGACGCGCTTTTACTGC-3 ), 5 L 10× Ex Taq buffer, 4 L dNTP mixture, 3 L cooling E. coli cell lysate (E. coli cell boiled at 95 ◦ C for 10 min), 35.5 L distilled water and 0.5 Ex Taq DNA polymerase. The aroGM gene was amplified from MG1655 DNA using the same reaction conditions but the aroGM12 and aroGM34 fragment was substitute for template and the same thermal program except for the extension time of 1.5 min at 72 ◦ C. The prime for aroGM was 5 -CCATGGGCAATTATCAGAACGACGATTTA3 and 5 -GGATCCCGCCCGCGACGCGCTTTTACTGC-3 . 2.3.3. The transform and extraction of plasmid pGM-T-aroGM After gel purification, aroGM gene fragment was ligated into pGM-T Vector at 16 ◦ C for 6 h. And then, the mixture was transformed to competent cells of E. coli TOP 10 as follows: (1) The competent cells were taken from a −80 ◦ C freezer to an ice-bath until the competent cells were melted. (2) 10 L of the ligation mixture was added to the competent cell suspension and placed in an ice-bath for 30 min. (3) The centrifuge tube was placed in a 42 ◦ C water-bath for 90 s, and then quickly moved to an ice-bath for 2 min. (4) 200 L germfree LB medium was added to the tube and the mixture was at 37 ◦ C in a thermostatic oscillation incubator for 45 min. (5) 200 L competent cell were transferred to the LB plate (which contained added ampicillin antibiotics) and was spread evenly; the plate was placed upside down when the liquid was fully absorbed and incubate it at 37 ◦ C for 14 h. Plasmid DNA was extracted from E. coli using the Biopin plasmid DNA Extraction Kit (BioFlux, China), the improvement procedure was as follows: (1) Add 1.5 mL cultured bacteria to a 1.5 mL micro centrifuge tube. (2) Centrifuge at 10,000 rpm for 30 s, and discard the supernatant. In order to increase the concentration of plasmid DNA, repeat steps one and two. (3) Resuspend pelleted bacterial cells in 300 L resuspension buffer and no cell clumps should be visible after resuspension of the pellets. (4) Add 300 L lysis buffer
S. Lin et al. / International Journal of Biological Macromolecules 51 (2012) 915–919
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Table 2 The reaction system of double digests. Components
Volume/L
pGM-T-aroGM Water, nuclease-free 10× FastDigest buffer F.D. NcoI F.D. BamHI Total
5 3 1 0.5 0.5 10
and gently invert the tube 4–6 times to mix. (5) Add 400 L neutralization buffer and gently invert the tube 4–6 times to mix. (6) Centrifuge for 15 min at 13,000 rpm until a compact white pellet forms. (7) Apply the supernatant to the spin column and centrifuge for 30–60 s at 6000 × g, discard the flow-through. (8) Add 650 L wash buffer to the spin column and centrifuge for 30–60 s at 12,000 × g, discard the flow-through. (9) Repeat step 8 once. (10) Centrifuge for an additional 1 min at 12,000 × g and transfer the spin column to a sterile 1.5 mL micro centrifuge tube, until wash buffer has completely volatilized. (11) Add 50 L elution buffer (preheat elution buffer with a 50 ◦ C water bath) to the spin column and let it stand for 1 min at room temperature. Centrifuge for 1 min at 12,000 × g. The buffer in the micro centrifuge tube contains the plasmid DNA, and then the buffer contains plasmid DNA was added the Spin Column again centrifuging to improve recovery efficiency of plasmid DNA. Then the plasmids pGM-T-aroGM was incubated at 37 ◦ C with FastDigest NcoI and FastDigest BamHI (FastDigest enzymes are an advanced line of restriction enzymes for rapid DNA digestion) for 15 min, the reaction conditions are listed in Table 2. The double digested production of plasmid pGM-T-aroGM was identification using 0.8% agarose gel electrophoresis and scan. The positive clones which contained target gene were verified by nucleotide sequencing. 2.3.4. The construction of pET-28b (+)-aroGM vector Prokaryotic expression vector pET-28b (+) was used for the expression of target genes. Multiple cloning sites that the vector contains are preceded by a T7 promoter. The vector also carries the kanamycin resistance gene. In the work, the NcoI–BamHI fragment of pET-28b (+) was deleted and the rest was purified from agarose gel by using gel mini purification kit (TIANGEN, China). At the same time, the NcoI–BamHI fragment of pGMT-aroGM was extracted. After gel purification, the aroGM was subcloned into the NcoI and the BamHI site of pET-28b (+) to create the plasmids pET-28b (+)-aroGM. Recombinant plasmids were analyzed by restriction digestion and verified by nucleotide sequencing.
Fig. 1. The electrophoresis result of aroGM12 amplification (from left to right: 1: DNA D2000, 2–4: PCR production of aroGM12).
Fig. 2. The electrophoresis result of aroGM34 amplification (from left to right: 1: DNA D2000, 2, 3: PCR production of aroGM34).
destained over-night in destaining solution (ethanol 250 mL, acetic acid 70 mL, double distilled water 380 mL) and scanned. 3. Results and discussion 3.1. The cloning of aroGM gene To determine the effects of aroG mutation on the protein expression, aroG mutation gene was cloned. The PCR fragment about 525 bp and 528 bp is shown in Figs. 1 and 2, respectively. The DNA lengths were similar to aroGM12 and aroGM34 fragments. The aroGM gene was cloned using splicing overlap extension PCR (SOE-PCR) technique, through analyzing the electrophoresis result of aroGM known, the DNA fragment in Fig. 3 is about 1000 bp, and this result is consistent with the length of aroGM.
2.3.5. The induction of E. coli BL21 (DE3) engineering bacteria The strains E. coli BL21 (DE3) contained the plasmid pET-28b (+) and pET-28b (+)-aroGM were propagated at 37 ◦ C in germfree LB medium containing 50 g kanamycin/mL. Overnight cultures were diluted 1/100 into the same medium, grown to OD600 of 0.7–0.9, E. coli BL21 (DE3) harboring pET-28b (+)-aroGM was induced with 1 mM isopropyl--d-thiogalactopyranoside (IPTG), and cultivated for an additional 5 h as well as E. coli BL21 (DE3) harboring pET-28b (+). 2.3.6. SDS-PAGE analysis SDS-PAGE was run according to Laemmli [20] on the MiniProtean 4 gel system (BioRad, Richmond, CA, USA). SDS-PAGE was performed in 12% acrylamide gels and the proteins were visualized by staining with Coomassie Brilliant Blue R-250 (CBB-R250 0.5 g, carbinol 125 mL, acetic acid 35 mL, added distilled water to 500 mL),
Fig. 3. The electrophoresis result of aroGM amplification (from left to right: 1: Maker III, 2–4: PCR production of aroGM).
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Fig. 4. Sketch of expression vector pET-28b (+)-aroGM.
Fig. 6. SDS-PAGE analysis of the proteins of the E. coli BL21 (DE3) (from left to right: lanes 1 and 4: protein size Maker MW marker included (up to down) -galactosidase: 118 kDa; bovine serum albumin: 90 kDa; ovalbumin: 50 kDa; carbonic anhydrase: 36 kDa; -lactoglobulin: 27 kDa; lysozyme: 20 kDa; lane 2: solution protein of aroGM gene expression; lane 3: solution protein expression level of strains harbored pET-28b (+); lane 5: inclusion body protein of aroGM gene expression; lane 6: inclusion body protein expression level of strains harbored pET-28b (+)).
3.2. The construction of pGM-T-aroGM vector The aroGM fragment was integrated into pGM-T vector. The plasmid pGM-T-aroGM was incubated by the FastDigest NcoI and FastDigest BamHI at 37 ◦ C. The nine different plasmids were digested; there are two different size fragments of 1053 bp and 3015 bp, the lengths were similar to the size of aroGM and linear pGM-T. The pGM-T-aroGM strains were verified by sequencing. The DNA sequencing result of vector pGM-T-aroGM showed that the residue Leu175 of aroG was successfully replaced with Asp. 3.3. The construction of aroGM expression vector The aroGM gene under control of T7 promoter was cloned into expression vector next to pET-28b (+), resulting the plasmid pET-28b (+)-aroGM. The construction sketch of expression vector pET-28b (+)-aroGM is shown in Fig. 4. After transforming and plasmid extracting, the recombination plasmid pET-28b (+)-aroGM was digested by the FastDigest NcoI and FastDigest BamHI. The size of fragments is similar to aroGM and linear pET-28b (+) (Fig. 5). This conclusion indicated that the plasmid construction is successful. 3.4. The result of SDS-PAGE analysis Induced bacterial cells were harvested by centrifugation, resuspended in 1/20 volume the GuNTA-0 buffer (20 mM Tris–HCl, pH 7.9, 0.5 M NaCl, 10% glycerol, 6 M guanidium HCl, 1 mM PMSF) of cultured bacteria. The protein was released from cells by ultrasonic treatments (200 W, sonicated 3 s each 10 s) in ice for 30 min, and
then centrifuged for 15 min at 13,000 rpm at 4 ◦ C. The protein samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 2 mL 5% stacking gel which was used to concentrate protein is prepared with 1.4 mL distilled water, 0.33 mL 30% Acr-Bis (29: 1), 0.25 mL 1 M Tris–HCl, pH 6.8, 10% SDS and 10% AP 0.02 mL, respectively, and 0.002 mL TEMED, added components from distilled water to TEMED in turn. The resolving gel consists of 1 mL distilled water, 2.0 mL 30% Acr-Bis (29: 1), 1.9 mL 1 M Tris–HCl, pH 8.8, 0.05 mL 10% SDS, 0.05 mL 10% AP and 0.002 mL TEMED. In this gel, macro molecules separate according to their size. The protein of strains harboring plasmid pET-28b (+) was the control group. SDS-PAGE showed the expression level of strain harbored the pET-28b (+)-aroGM plasmid and control group that only contained pET-28b (+) in the stain (Fig. 6). Lanes 1 and 4 are molecular weight markers. Compared samples from E. coli BL21 (DE3) harboring pET-28b (+)-aroGM (lane 2) with samples from E. coli BL21 (DE3) harboring pET-28b (+) (lane 3), the solution protein of the two strains have no intense change; however the inclusion body protein quantity from the two strains was obviously different. The prominent band is at approximately 41 kDa in lane 5, but it was not observed in the control group (lane 6). It is considered that the mutation caused the over-expression of aroG gene. 4. Conclusions The effect of site-directed mutational aroG gene on the expression level of aroG gene was investigated. The expression level of test and control groups was evaluated by SDS-PAGE. Based on the polymerase chain reaction technique, splicing overlap extension PCR (SOE-PCR) was used to achieve site-directed mutagenesis of aroG gene. The residue Leu175 of aroG was successfully replaced with Asp. The recombinant DNA technology was used in the construction of plasmids pGM-T-aroGM and pET-28b (+)-aroGM. SDS-PAGE was used to access the expression level of strain harbored recombinant plasmid. In conclusion, the mutation of residue Leu175 of aroG DNA sequences significantly achieved the over-expression of aroG gene. Further work is needed to estimate quantitatively protein expression level of strain contained plasmid pET-28b (+)-aroGM by western blotting. Acknowledgement
Fig. 5. Enzymatic result of pET-28b (+)-aroGM (1: D15000; 2, 4: F.D. NcoI and BamHI digest production of plasmids 3, 5).
The authors acknowledge the financial support provided by the Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-Year Plan Period (2012BAD33B03).
S. Lin et al. / International Journal of Biological Macromolecules 51 (2012) 915–919
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Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Site-directed mutagenesis and over expression of aroG gene of Escherichia coli K-12 Songyi Lin a,b,1 , Xiujuan Meng a,1 , Jie Jiang a,1 , Daxing Pang b , Gregory Jones c , Hongsheng OuYang b,∗ , Linzhu Ren b,∗∗ a
Laboratory of Nutrition and Functional Food, College of Quartermaster Technology, Jilin University, Changchun 130062, PR China Post-doctoral Research Station of Biology, Jilin University, Changchun 130062, PR China c Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC 29634, USA b
a r t i c l e
i n f o
Article history: Received 25 June 2012 Accepted 11 July 2012 Available online 20 July 2012 Keywords: aroG Site-directed mutagenesis Over-expression
a b s t r a c t 3-Deoxy-d-arabino-heptulonate-7-phosphate (DAHP) synthetase is one of the key enzymes, which catalyzes the first step in the aromatic amino acid biosynthetic pathway and yields the three amino acids tyrosine, tryptophan, and phenylalanine. In Escherichia coli (E. coli), three differently regulated DAHP synthases carry out the first regulated step in the aromatic amino acid biosynthetic pathway. The three DAHP synthases encoded by the genes aroG, aroF and aroH are inhibited by phenylalanine, tyrosine and tryptophan, respectively. In this work, the aroG gene was cloned and mutated by site-directed mutagenesis using splicing overlap extension PCR (SOE-PCR) technique. The feedback-resistant DAHP synthase encoded by aroG was achieved by replacing the residue Leu175 of aroG with Asp as to increase net carbon flow down the common pathway. SDS-PAGE which was used to access the protein expression level of aroGM showed the strain harbored mutated aroGM gene achieve over-expression compared to strain contain empty plasmid pET-28b (+). © 2012 Elsevier B.V. All rights reserved.
1. Introduction The aromatic amino acids, l-tryptophan, l-phenylalanine, and l-tyrosine, can be manufactured by bacterial fermentation. lTryptophan is an essential amino acid that is required for the biosynthesis of proteins and is the precursor for several important biological compounds [1,2]. l-Tryptophan is also an important
Abbreviations: PEP, phosphoenolpyruvate; E4P, erythrose 4-phosphate; DAHP, 3-deoxy-d-arabino-heptulosonate-7-phosphate; PCR, polymerase chain reaction; SOE-PCR, splicing overlap extension polymerase chain reaction; LB, Luria Bertani; IPTG, isopropyl--d-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; AP, ammonium persulfate; TEMED, N,N,N ,N tetramethylethylenediamine; PMSF, phenylmethanesulfonyl fluoride. ∗ Corresponding author. Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi’an Road, Changchun 130062, PR China. Tel.: +86 131 59680552; fax: +86 431 86758018. ∗∗ Corresponding author. Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi’an Road, Changchun 130062, PR China. Tel.: +86 431 87836175; fax: +86 431 86758018. E-mail addresses: ouyanghongsheng [email protected] (H. OuYang), [email protected] (L. Ren). 1 These authors contributed equally to this study and should be regarded as first joint author. 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.07.013
amino acid in terms of its applications in the pharmaceutical, food, and cosmetics industries [3,4]. l-Tryptophan serves as a medicine to improve sleep state, mood and is a food additive. In addition, other industrially relevant substances can be derived from tryptophan synthesis intermediates, like indigo [5] or the anti-influenza drug Oseltamivir phosphate [6]. Chemical synthesis, enzymatic/microbial conversion and microbial fermentation are the methods used for industrial production of l-tryptophan. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to strain improvement. The first application of recombinant DNA technology to strain improvement for l-tryptophan production was performed with Escherichia coli by Tribe and Pittard [7]. They reported increased production by amplification of the trp operon with a deregulated trpE gene, though the final titer of about 1 g/L was low. On the other hand, Aiba et al. constructed a genetically engineered strain by introducing a plasmid containing the trp operon with deregulated trpE and trpD genes into an E. coli trpR and tnaA mutant [8]. The feedback-resistant anthranilate (ANTA) synthase encoded by trpED was achieved by replacing the residue Ser40 of TrpE with Phe [9,10]. The biosynthesis of l-tryptophan is initiated by the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP). 3Deoxy-d-arabino-heptulonate-7-phosphate synthetase (DAHPS) is
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one of the key enzymes, which catalyzes the first step in the aromatic amino acid biosynthetic pathway and yields the three amino acids tyrosine, tryptophan, and phenylalanine. In E. coli, there are three DAHPS isoforms, each specifically inhibited by one of the three aromatic amino acids [11,12]. DAHPS (Phe), a homotetramer encoded by the aroG gene [13], is the major isoform, constituting about 80% of the total activity of the cell [14–16]. The less abundant isozymes, DAHPS (Trp) (1% of total activity) and DAHPS (Tyr) (20% of total activity) are homodimers encoded by the aroH and aroF genes, respectively [17]. 3-Deoxy-d-arabino-heptulosonate-7-phosphate is catalyzed to generate chorismate through three reactions, and then the l-tryptophan branch pathway leading to l-tryptophan. The feedback inhibition site of aroG gene consists of Asp6, Asp7, Ile10, Ilel3, Prol50, Gin151, Leul75, Leul79, Ser180, Phe209, Ser211 and Val221. The replacement of residue Leu175 of aroG with Asp generated phenylalanine-insensitive form of AroG and showed a higher DAHPS activity in the presence of 1 mM phenylalanine compared to the wide-type DAHPS [18]. Based on the previous study, we attempted to introduce defined genetic manipulations into E. coli to develop an l-Trp-producing strain based on known regulatory and metabolic information. The feedback-resistant DAHP synthase encoded by aroG was achieved by replacing the residue Leu175 of aroG with Asp as to increase net carbon flow down the chorismate and more importantly is to increase l-tryptophan production. The feedback-resistant aroG genes was named aroGM and was assembled on the vectors pET-28b (+) and realized over-expressed in E. coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used in the study to test the expression of protein. 2. Materials and methods 2.1. Bacterial strains and plasmids The genome of wild-type E. coli K-12 was used as template to obtain the aroG using polymerase chain reaction (PCR) technology. The pET-28b (+) vectors which were modified as expression vector carry an N-terminal His·Tag/thrombin/T7·Tag configuration plus an optional C-terminal His·Tag sequence. Bacterial strains and plasmids used in this study are listed in Table 1. Strains and relevant details of their properties is also described. 2.2. Media and manipulations The Ex Taq DNA polymerase, T4 DNA ligase and restriction endonucleases were purchased from MBI Fermentas (Burlington, ON, Canada). The pGM-T vector was purchased from TIANGEN (Beijing, China). All enzymatic reactions were performed according to Table 1 Bacterial strains and plasmids used in this study. Strains/plasmids E. coli Escherichia coli k-12 E. coli BL21 (DE3) E. coli Top10
Plasmid pGM-T pET-28b (+) pET-28b (+)-aroGM
Relevant characteristics
Source Lab. save
−
−
ompT, hsdSB (rB mB ), gal dcm(DE3) mcrA (mrr-hsdRMS-mcrBC) 80 lacZM15lacX74 recA1 ara 139 (ara-leu) 7697galU galK rpsL (Strr) endA1 nupG
TIANGEN
Ampr Kanr Contains the aroGM gene
TIANGEN Lab. save This work
TIANGEN
the manufacturers’ instructions. E. coli cells were transformed by heat shock using frozen competent cells prepared using the calcium chloride method described in Molecular Cloning [19]. Plasmid DNA was extracted from E. coli using the Biopin plasmid DNA Extraction Kit (BioFlux, China). Primer Premier 5.0 was used to design primer and DNAMAN was used to achieve sequence alignment. The E. coli strains its derivatives were grown aerobically on a shaker at 37 ◦ C for 12 h in Luria Bertani (LB) medium which contained 10 g/L trypeptone; 10 g/L NaCl; 5 g/L yeast extract or on LB plates which contained10 g/L trypeptone; 10 g/L NaCl; 5 g/L yeast extract and 15 g/L agar powder. When required, 100 g ampicillin/mL or kanamycin was added. 2.3. Cloning and expression of aroGM 2.3.1. Nucleotide sequence accession numbers The complete genome sequences of E. coli str. K-12 substr. MG1655 studied in this paper are available from NCBI under accession number U00096, the sequences of the aroG gene are deposited in NCBI under version numbers GI: 48994873. 2.3.2. The cloning of aroGM gene The aroGM12 (1–525 bp) and aroGM34 (525–1053 bp) genes of E. coli MG1655 were amplified by splicing overlap extension polymerase chain reaction (SOE-PCR) in Thermal Cycler (Thermo Electron Corporation, Milford, MA, USA) with the thermal program of 1 cycle of 94 ◦ C for 5 min, 30 cycles of 94 ◦ C for 1 min, 55 ◦ C for 30 s, 72 ◦ C for 50 s, and 1 cycle of 72 ◦ C for 10 min. Fifty microliter of each reaction mixture contained 1 L each forward and reverse primers (aroGM12 with 5 -CCATGGTAGGATGCTCCTGTTATGGTCGTTATGTCGG-3 and 5 -AGCCCTGATGCATCTTCGCGGTGCAC-3 , and aroGM34 with 5 -GTGCACCGCGAAGATGCATCAGGGCT-3 and 5 GGATCCTTACCCGCGACGCGCTTTTACTGC-3 ), 5 L 10× Ex Taq buffer, 4 L dNTP mixture, 3 L cooling E. coli cell lysate (E. coli cell boiled at 95 ◦ C for 10 min), 35.5 L distilled water and 0.5 Ex Taq DNA polymerase. The aroGM gene was amplified from MG1655 DNA using the same reaction conditions but the aroGM12 and aroGM34 fragment was substitute for template and the same thermal program except for the extension time of 1.5 min at 72 ◦ C. The prime for aroGM was 5 -CCATGGGCAATTATCAGAACGACGATTTA3 and 5 -GGATCCCGCCCGCGACGCGCTTTTACTGC-3 . 2.3.3. The transform and extraction of plasmid pGM-T-aroGM After gel purification, aroGM gene fragment was ligated into pGM-T Vector at 16 ◦ C for 6 h. And then, the mixture was transformed to competent cells of E. coli TOP 10 as follows: (1) The competent cells were taken from a −80 ◦ C freezer to an ice-bath until the competent cells were melted. (2) 10 L of the ligation mixture was added to the competent cell suspension and placed in an ice-bath for 30 min. (3) The centrifuge tube was placed in a 42 ◦ C water-bath for 90 s, and then quickly moved to an ice-bath for 2 min. (4) 200 L germfree LB medium was added to the tube and the mixture was at 37 ◦ C in a thermostatic oscillation incubator for 45 min. (5) 200 L competent cell were transferred to the LB plate (which contained added ampicillin antibiotics) and was spread evenly; the plate was placed upside down when the liquid was fully absorbed and incubate it at 37 ◦ C for 14 h. Plasmid DNA was extracted from E. coli using the Biopin plasmid DNA Extraction Kit (BioFlux, China), the improvement procedure was as follows: (1) Add 1.5 mL cultured bacteria to a 1.5 mL micro centrifuge tube. (2) Centrifuge at 10,000 rpm for 30 s, and discard the supernatant. In order to increase the concentration of plasmid DNA, repeat steps one and two. (3) Resuspend pelleted bacterial cells in 300 L resuspension buffer and no cell clumps should be visible after resuspension of the pellets. (4) Add 300 L lysis buffer
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Table 2 The reaction system of double digests. Components
Volume/L
pGM-T-aroGM Water, nuclease-free 10× FastDigest buffer F.D. NcoI F.D. BamHI Total
5 3 1 0.5 0.5 10
and gently invert the tube 4–6 times to mix. (5) Add 400 L neutralization buffer and gently invert the tube 4–6 times to mix. (6) Centrifuge for 15 min at 13,000 rpm until a compact white pellet forms. (7) Apply the supernatant to the spin column and centrifuge for 30–60 s at 6000 × g, discard the flow-through. (8) Add 650 L wash buffer to the spin column and centrifuge for 30–60 s at 12,000 × g, discard the flow-through. (9) Repeat step 8 once. (10) Centrifuge for an additional 1 min at 12,000 × g and transfer the spin column to a sterile 1.5 mL micro centrifuge tube, until wash buffer has completely volatilized. (11) Add 50 L elution buffer (preheat elution buffer with a 50 ◦ C water bath) to the spin column and let it stand for 1 min at room temperature. Centrifuge for 1 min at 12,000 × g. The buffer in the micro centrifuge tube contains the plasmid DNA, and then the buffer contains plasmid DNA was added the Spin Column again centrifuging to improve recovery efficiency of plasmid DNA. Then the plasmids pGM-T-aroGM was incubated at 37 ◦ C with FastDigest NcoI and FastDigest BamHI (FastDigest enzymes are an advanced line of restriction enzymes for rapid DNA digestion) for 15 min, the reaction conditions are listed in Table 2. The double digested production of plasmid pGM-T-aroGM was identification using 0.8% agarose gel electrophoresis and scan. The positive clones which contained target gene were verified by nucleotide sequencing. 2.3.4. The construction of pET-28b (+)-aroGM vector Prokaryotic expression vector pET-28b (+) was used for the expression of target genes. Multiple cloning sites that the vector contains are preceded by a T7 promoter. The vector also carries the kanamycin resistance gene. In the work, the NcoI–BamHI fragment of pET-28b (+) was deleted and the rest was purified from agarose gel by using gel mini purification kit (TIANGEN, China). At the same time, the NcoI–BamHI fragment of pGMT-aroGM was extracted. After gel purification, the aroGM was subcloned into the NcoI and the BamHI site of pET-28b (+) to create the plasmids pET-28b (+)-aroGM. Recombinant plasmids were analyzed by restriction digestion and verified by nucleotide sequencing.
Fig. 1. The electrophoresis result of aroGM12 amplification (from left to right: 1: DNA D2000, 2–4: PCR production of aroGM12).
Fig. 2. The electrophoresis result of aroGM34 amplification (from left to right: 1: DNA D2000, 2, 3: PCR production of aroGM34).
destained over-night in destaining solution (ethanol 250 mL, acetic acid 70 mL, double distilled water 380 mL) and scanned. 3. Results and discussion 3.1. The cloning of aroGM gene To determine the effects of aroG mutation on the protein expression, aroG mutation gene was cloned. The PCR fragment about 525 bp and 528 bp is shown in Figs. 1 and 2, respectively. The DNA lengths were similar to aroGM12 and aroGM34 fragments. The aroGM gene was cloned using splicing overlap extension PCR (SOE-PCR) technique, through analyzing the electrophoresis result of aroGM known, the DNA fragment in Fig. 3 is about 1000 bp, and this result is consistent with the length of aroGM.
2.3.5. The induction of E. coli BL21 (DE3) engineering bacteria The strains E. coli BL21 (DE3) contained the plasmid pET-28b (+) and pET-28b (+)-aroGM were propagated at 37 ◦ C in germfree LB medium containing 50 g kanamycin/mL. Overnight cultures were diluted 1/100 into the same medium, grown to OD600 of 0.7–0.9, E. coli BL21 (DE3) harboring pET-28b (+)-aroGM was induced with 1 mM isopropyl--d-thiogalactopyranoside (IPTG), and cultivated for an additional 5 h as well as E. coli BL21 (DE3) harboring pET-28b (+). 2.3.6. SDS-PAGE analysis SDS-PAGE was run according to Laemmli [20] on the MiniProtean 4 gel system (BioRad, Richmond, CA, USA). SDS-PAGE was performed in 12% acrylamide gels and the proteins were visualized by staining with Coomassie Brilliant Blue R-250 (CBB-R250 0.5 g, carbinol 125 mL, acetic acid 35 mL, added distilled water to 500 mL),
Fig. 3. The electrophoresis result of aroGM amplification (from left to right: 1: Maker III, 2–4: PCR production of aroGM).
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Fig. 4. Sketch of expression vector pET-28b (+)-aroGM.
Fig. 6. SDS-PAGE analysis of the proteins of the E. coli BL21 (DE3) (from left to right: lanes 1 and 4: protein size Maker MW marker included (up to down) -galactosidase: 118 kDa; bovine serum albumin: 90 kDa; ovalbumin: 50 kDa; carbonic anhydrase: 36 kDa; -lactoglobulin: 27 kDa; lysozyme: 20 kDa; lane 2: solution protein of aroGM gene expression; lane 3: solution protein expression level of strains harbored pET-28b (+); lane 5: inclusion body protein of aroGM gene expression; lane 6: inclusion body protein expression level of strains harbored pET-28b (+)).
3.2. The construction of pGM-T-aroGM vector The aroGM fragment was integrated into pGM-T vector. The plasmid pGM-T-aroGM was incubated by the FastDigest NcoI and FastDigest BamHI at 37 ◦ C. The nine different plasmids were digested; there are two different size fragments of 1053 bp and 3015 bp, the lengths were similar to the size of aroGM and linear pGM-T. The pGM-T-aroGM strains were verified by sequencing. The DNA sequencing result of vector pGM-T-aroGM showed that the residue Leu175 of aroG was successfully replaced with Asp. 3.3. The construction of aroGM expression vector The aroGM gene under control of T7 promoter was cloned into expression vector next to pET-28b (+), resulting the plasmid pET-28b (+)-aroGM. The construction sketch of expression vector pET-28b (+)-aroGM is shown in Fig. 4. After transforming and plasmid extracting, the recombination plasmid pET-28b (+)-aroGM was digested by the FastDigest NcoI and FastDigest BamHI. The size of fragments is similar to aroGM and linear pET-28b (+) (Fig. 5). This conclusion indicated that the plasmid construction is successful. 3.4. The result of SDS-PAGE analysis Induced bacterial cells were harvested by centrifugation, resuspended in 1/20 volume the GuNTA-0 buffer (20 mM Tris–HCl, pH 7.9, 0.5 M NaCl, 10% glycerol, 6 M guanidium HCl, 1 mM PMSF) of cultured bacteria. The protein was released from cells by ultrasonic treatments (200 W, sonicated 3 s each 10 s) in ice for 30 min, and
then centrifuged for 15 min at 13,000 rpm at 4 ◦ C. The protein samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 2 mL 5% stacking gel which was used to concentrate protein is prepared with 1.4 mL distilled water, 0.33 mL 30% Acr-Bis (29: 1), 0.25 mL 1 M Tris–HCl, pH 6.8, 10% SDS and 10% AP 0.02 mL, respectively, and 0.002 mL TEMED, added components from distilled water to TEMED in turn. The resolving gel consists of 1 mL distilled water, 2.0 mL 30% Acr-Bis (29: 1), 1.9 mL 1 M Tris–HCl, pH 8.8, 0.05 mL 10% SDS, 0.05 mL 10% AP and 0.002 mL TEMED. In this gel, macro molecules separate according to their size. The protein of strains harboring plasmid pET-28b (+) was the control group. SDS-PAGE showed the expression level of strain harbored the pET-28b (+)-aroGM plasmid and control group that only contained pET-28b (+) in the stain (Fig. 6). Lanes 1 and 4 are molecular weight markers. Compared samples from E. coli BL21 (DE3) harboring pET-28b (+)-aroGM (lane 2) with samples from E. coli BL21 (DE3) harboring pET-28b (+) (lane 3), the solution protein of the two strains have no intense change; however the inclusion body protein quantity from the two strains was obviously different. The prominent band is at approximately 41 kDa in lane 5, but it was not observed in the control group (lane 6). It is considered that the mutation caused the over-expression of aroG gene. 4. Conclusions The effect of site-directed mutational aroG gene on the expression level of aroG gene was investigated. The expression level of test and control groups was evaluated by SDS-PAGE. Based on the polymerase chain reaction technique, splicing overlap extension PCR (SOE-PCR) was used to achieve site-directed mutagenesis of aroG gene. The residue Leu175 of aroG was successfully replaced with Asp. The recombinant DNA technology was used in the construction of plasmids pGM-T-aroGM and pET-28b (+)-aroGM. SDS-PAGE was used to access the expression level of strain harbored recombinant plasmid. In conclusion, the mutation of residue Leu175 of aroG DNA sequences significantly achieved the over-expression of aroG gene. Further work is needed to estimate quantitatively protein expression level of strain contained plasmid pET-28b (+)-aroGM by western blotting. Acknowledgement
Fig. 5. Enzymatic result of pET-28b (+)-aroGM (1: D15000; 2, 4: F.D. NcoI and BamHI digest production of plasmids 3, 5).
The authors acknowledge the financial support provided by the Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-Year Plan Period (2012BAD33B03).
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