Cloning and expression of the genes encoding glycerol dehydratase reactivase and identification of its biological activity

CHINESE JOURNAL OF BIOTECHNOLOGY Volume 22, Issue 6, November 2006 Online English edition of the Chinese language journal Cite this article as: Chin J Biotech, 2006, 22(6), 950−955.

RESEARCH PAPER

Cloning and Expression of the Genes Encoding Glycerol Dehydratase Reactivase and Identification of Its Biological Activity LI Wen-Jun, FANG Bai-Shan*, HONG Yan, WANG Xiao-Xia, LIN Jin-Xia, LIU Gui-Lan Key Laboratory of Industrial Biotechnology, Hua Qiao University), Quanzhou 362021, China

Abstract: The gdrA and gdrB genes encoding glycerol dehydratase reactivase were amplified using the genomic DNA of Klebsiella pneumoniae as the template. The gdrA and gdrB genes were inserted into pMD-18T to yield the recombinant cloning vector pMD-gdrAB. After the DNA sequence was determined, the gdrAB gene was subcloned into expression vector pET-28a(+) to yield the recombinant expression vector pET-28gdrAB. Under screening pressure by ampicillin and kanamycin simultaneously, the activity of glycerol dehydratase reactivase was characterized by the coexpression of pET-32gldABC, which carried the gldABC gene encoding glycerol dehydratase, and pET-28gdrAB in E. coli BL21 (DE3). Key Words:

glycerol dehydratase; glycerol dehydratase reactivase; coexpression; incompatible plasmids; molecular chaperone

3-Hydroxypropionaldehyde (3-HPA) has several significant industrial applications. It can be used as a food preservative, as a precursor for the synthesis of many modern chemicals such as acrolein, acrylic acid, and 1,3-propanediol (1,3-PD), and for the synthesis of polymers[1,2]. To date, 3-HPA can be mainly obtained through hydration technology of acrolein[3] and hydroformylation of ethylene oxide[4]; however, these techniques have several disadvantages. Nowadays, the microbiological fermentation method for yielding 3-HPA has attracted the attention of several researchers. Glycerol dehydratase (GDHt; EC 4.2.1.30) catalyzes the rate-limiting step in the anaerobic conversion of glycerol to 1,3-PD. During the coenzyme B12-dependent catalysis, the enzyme undergoes irreversible inactivation by glycerol or oxygen. Inactivation involves irreversible cleavage of the Co–C bond of coenzyme B12, forming 5′-deoxyadenosine and an alkylcobalamin-like species, which tightly bind to the

GDHt’s active site, thereby resulting in severe limitation of the organism’s ability to ferment glycerol[5]. However, the recent studies have indicated that two homologous open reading frames (gdrA and gdrB) for the GDHt of Klebsiella pneumoniae were proposed to be involved in the reactivation of GDHt. They named the gene products glycerol dehydratase reactivase, which rapidly reactivated glycerol-inactivated or O2-inactivated GDHt in the presence of ATP, Mg2+, and coenzyme B12[6–9]. According to the analysis of the crystal structure of reactivase, Liao DI proposed ‘subunit swap’ hypothesis to interpret the mechanism of reactivation[10]. The glycerol dehydratase reactivase has been an attractive component for the study on microbiological fermentation. Generally, two incompatible plasmids cannot be used in the coexpression of recombinant protein in E. coli because of their rapid loss during cell growth and distribution[11]. However, this drawback can be overcome when two kinds of antibiotic

Received: June 27, 2006; Accepted: July 19, 2006. * Corresponding author. Tel: +86-595-2691560; E-mail: [email protected] This work was financially supported by the National Natural Science Foundation of China (Nos. 20276026, 20446004), the Science and Technology Foundation of Fujian Province, China (No.20031020), and the Natural Science Foundation of HuaQiao University, China (No. 03HZR2). Copyright © 2006, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.

LI Wen-Jun et al. / Chinese Journal of Biotechnology, 2006, 22(6): 950–955

are present in the selective medium[12,13]. In the present study, the gdrA and gdrB genes have been amplified using the genomic DNA of Klebsiella pneumoniae as the template, and the expression plasmid has been constructed. Moreover, the activity of glycerol dehydratase reactivase was characterized by coexpression of two incompatible plasmids in E. coli BL21 (DE3). The use of the two incompatible plasmids in the coexpression of recombinant protein in E. coli under screening pressure by ampicillin and kanamycin simultaneously has been approved.

1

Materials and methods

1.1 Materials 1.1.1 Bacterial strains and plasmids: The overexpression vector, pET-32gldABC, which was constructed in our laboratory , was used for the expression of the gldABC (GDHt) genes. pMD18-T and pET-28a(+) were preserved in our laboratory. E. coli DH5α and E. coli BL21 (DE3) were used as hosts for the cloning and expression experiments. Klebsiella pneumoniae DSM2026 was provided by Dr. Zheng AP (German Research Center for Biotechnology). 1.1.2 Enzyme tool and reagents: T4 DNA ligase, restriction endonucleases, and rTaq DNA polymerase were obtained from TaKaRa Biotechnology (Dalian, China) Co Ltd. E.Z.N.A. Gel extraction Kit, E.Z.N.A. Plasmid Miniprep Kit and ATP were provided by Xiamen Tagene Biotechnology Ltd, China. Coenzyme B12 and 3-methyl-2-benzothiazolinone hydrazone (MBTH) were from SIGMA Corporation, USA. Other chemical reagents were analytically pure and were purchased from China. 1.1.3 Media and growth conditions: E. coli was routinely grown at 37 °C in Luria-Bertani medium, supplemented with ampicillin (100 mg/L) and kanamycin (50 mg/L) whenever necessary. 1.1.4 Primers (synthesized by Peking AuGCT Biotechnology Ltd, China): According to the sequences of dhaB4 and orf2b from K. pneumoniae (ATCC25955) (GenBank accession number U30903), two pairs of primers were designed in this study. Primers for amplifying gdrA were as follows (without restriction recognition sites but including ribosome recombinant site): Primer I: 5′-ATGCGGAGGTCAGCATGCCGTTAATAG-3′; Primer II: 5′-AGATTAGCCTGACCAGCCAGTAGCAGC-3′; Primers for amplifying gdrB were (including EcoR I and Sma I respectively): Primer III: 5′-CCGGAATTCTCGCTTTCACCGCCA-3′; Primer IV: 5′-TCCCCCGGGTCAATTTCTCTCACT-3′. B

1.2 Methods 1.2.1 PCR amplifying gdrA and gdrB: The PCR programs

for amplifying gdrA was: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 50 s, annealing at 57 °C for 45 s, extension at 72 °C for 2 min (30 cycles), and final extension at 72 °C for 10 min. Programs for amplifying gdrB was: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 50 s, annealing at 55 °C for 45 s, extension at 72 °C for 50 s (30 cycles), and final extension at 72 °C for 10 min. 1.2.2 Construction of the cloning vector pMD-gdrAB: GdrA was cloned into pMD-18T vector to construct pMD-gdrA by TA cloning. GdrB was double digested with EcoR I and Sma I, and inserted into pMD-gdrA, which had been linearized with the same enzymes to construct the cloning vector pMDgdrAB. The positive clones identified by restriction enzyme digestion analysis were subjected to DNA sequence analysis (AuGCT Biotechnology Ltd, Peking, China). 1.2.3 Construction of the expression vector pET-28gdrAB: After pMD-gdrAB was double digested with EcoR I and Hind III, the gdrAB gene was isolated and inserted into pET-28a(+), which had been digested with the same enzymes. The positive clones identified by restriction enzyme digestion were transformed into host bacterium E. coli BL21 (DE3) for expression. 1.2.4 SDS-PAGE: Pellets of cells were suspended in 1× loading buffer and heated to 100 °C for 3 min and were analyzed using 15 % gels of SDS-PAGE electrophoresis with Coomassie Brilliant Blue R-250 stain. The expression products were compared with or without inducing IPTG in E .coli BL21 (DE3), respectively . 1.2.5 Expression of recombinant vectors in E. coli BL21 (DE3): Equal concentration of pET-28gdrAB and pET-32gldABC were cotransformed into E. coli BL21 (DE3). The recombinant E. coli was selected in Luria-Bertani medium containing 100 mg/L ampicillin and 50 mg/L kanamycin, and was then grown with appropriate antibiotics at 37 °C. A total of 1mmol/L IPTG was added to the culture when OD600 reached approximately 0.4. The cultures were incubated for 5 h at 25 °C, and then the cells were collected by centrifugation. 1.2.6 Aether treatment: Cells harvested in the late exponential phase were washed twice with 0.05 mol/L potassium phosphate buffer (pH 8.0) and suspended in the same buffer to a concentration of 0.1 g/mL. Aether (final concentration, 0.5 % [V/V]) was added into the cell suspension, and the mixture was vigorously shaken for 1 min at room temperature. The cells were then collected by centrifugation, washed twice with 0.05 mol/L potassium phosphate buffer (pH 8.0), and then suspended in the same buffer to a concentration of 0.05 g/mL. 1.2.7 Assays of GDHt and reactivase: The activity of the in situ GDHt was assayed by the MBTH method of Toraya et al[14]. This method was based on the ability of aldehydes to react with MBTH, forming the azine derivatives, which can be determined spectrophotometrically. The usual assay mixture

LI Wen-Jun et al. / Chinese Journal of Biotechnology, 2006, 22(6): 950–955

contained an appropriate amount of aether-treated cells, 0.2 mol/L glycerol, 0.05 mol/L KCl, 0.035 mol/L potassium phosphate buffer (pH 8.0), and 15 μmol/L coenzyme B12, in a total volume of 1 mL. After incubation at 37 °C for 10 min, the enzyme reaction was terminated by adding 1 mL of 0.1 mol/L potassium citrate buffer (pH 3.6) and 0.5 mL of 0.1 % MBTH hydrochloride. After 15 min at 37 °C, 1 mL of water was added and the amount of propionaldehyde was determined from the absorbance at 305 nm. Activity of glycerol dehydratase reactivase was determined using the same method with GDHt; however, a slight difference occurred in assay mixture, which included 0.2 mol/L glycerol, 0.05 mol/L KCl, 0.035 mol/L potassium phosphate buffer (pH 8.0), 15 μmol/L coenzyme B12, 3 mol/L ATP, and 3 mol/L MgCl2 in a total volume of 1 mL.

2

2.2 Construction and identification of cloning vector pMD-gdrAB The construction of cloning vector pMD-gdrAB is shown in Fig. 3. A band of 4 900 bp appeared when pMD-gdrAB was digested by EcoR I. Two segments of 2 700 bp and 2 200 bp were obtained when the recombinant vector was digested by EcoR I and Hind III. The results showed that fragments of appropriate length were obtained after digestion (Fig. 4).

Results

2.1 PCR amplification of gdrA and gdrB The PCR product of gdrA, approximately 1 840 bp including ribosomal recombinant site, was obtained as expected (Fig. 1). A 370-bp DNA fragment of the entire gdrB gene was correctly amplified from K. pneumoniae genome (Fig. 2).

Fig. 3 Construction of recombinant cloning vector pMD-gdrAB

Fig. 1 Electrophoresis of PCR products of gdrA M: DNA marker; 1: PCR products.

Fig. 4 Characterization of pMD-gdrAB digested by EcoRⅠand Hind Ⅲ/EcoRⅠ M: DNA marker; 1: pMD-gdrAB digested by EcoR I and Hind III; 2: pMD-gdrAB digested by EcoR I.

Fig. 2 Electrophoresis of PCR products of gdrB M: DNA marker; 1: PCR products.

2.3 Construction and identification of expression vector pET-28gdrAB The construction of expression vector pET-28gdrAB is shown in Fig. 5. When the expression vector was digested with EcoR I, a strip of 7 600 bp appeared. Two fragments of 3 100 bp and 4 500 bp were obtained on digestion with Sma I.

LI Wen-Jun et al. / Chinese Journal of Biotechnology, 2006, 22(6): 950–955

Upon cleaving the pET-28gdrAB with EcoR I and Hind III at the appropriate position, two strips of 5 400 bp and 2 200 bp would appear. All the results of the confirmation of restriction enzymes were consistent with the anticipated results (Fig. 6).

bp were obtained when the two recombinant expression plasmids were digested by EcoR I and Hind III. After cleavage by EcoR I, two fragments of 7 569 bp and 8 600 bp were formed. It had been confirmed that two incompatible plasmids were able to exit in E. coli BL21 (DE3) simultaneously.

Fig. 5 Construction of recombinant expression vector pET-28gdrAB

Fig. 7 Restriction analysis of the plasmids in E. coli BL21 cotransformants M: DNA marker; 1: pET-28gdrAB and pET-32gldABC digested by EcoR I and Hind III; 2: pET-28gdrAB and pET-32gldABC digested by EcoR I.

Fig. 6 Characterization of pET-28gdrAB digested by EcoR I and Hind III/Sma I/EcoR I M: DNA marker; 1: pET-28gdrAB digested by EcoR I and Hind III; 2: pET-28gdrAB digested by Sma I; 3: pET-28gdrAB digested by EcoR I.

2.4 Cotransformation of recombinant vectors into E. coli BL21 and restriction confirmation of endonucleases The recombinant E. coli was selected in LB medium containing 100 mg/L ampicillin and 50 mg/L kanamycin after equal quantities of recombinant vectors pET-28gdrAB and pET-32gldABC were cotransformed into E. coli BL21 (DE3). The positive E. coli recombinant was cultured overnight at 37 °C, and then plasmid DNA was extracted and identified by restriction enzymes (Fig. 7). For the existing two kinds of vectors, four strips of 2 200 bp, 2 700 bp, 5 369 bp, and 5 900

2.5 SDS-PAGE analysis of expressed protein in E. coli BL21 Recombinant E. coli BL21 (pET-28gdrAB), E. coli BL21 (pET-32gldABC), and E. coli BL21 (pET-28gdrAB, pET32gldABC) were selected from LB medium containing the corresponding antibiotic. Strains were cultured at 37 °C when OD600 approached 0.4, and then IPTG (final concentration 1mmol/L) was added to the culture. Having been incubated for 5 h at 25 °C, the recombinant cells were collected by centrifugation and were subjected to SDS-PAGE analysis (Fig. 8). Faint protein bands corresponding to the α and β subunits of glycerol dehydratase reactivase, with apparent molecular masses of 66 kD and 16 kD, were observed for the E. coli cells carrying pET-28gdrAB. Thick protein bands of 66 kD, 21 kD, and 16 kD corresponding to the α, β and γ subunits of GDHt were observed for E. coli cells containing pET32gldABC. Moreover, the protein bands of E. coli cells carrying pET-28gdrAB and pET-32gldABC appeared in the same position in the gel for E. coli cells with pET-32gldABC because the α and β subunits of glycerol dehydratase reactivase were almost similar to the α and γ subunits of GDHt.

LI Wen-Jun et al. / Chinese Journal of Biotechnology, 2006, 22(6): 950–955

2.6 Assays of glycerol dehydratase reactivase The recombinant E. coli containing pET-28gdrAB and pET-32gldABC was simultaneously cultured under screening pressure by ampicillin and kanamycin. Having been induced by IPTG, the recombinant cell was treated by aether for characterization of glycerol dehydratase reactivase (Fig. 9). When ATP and Mg2+ were absent in the assay mixture, the 3-HPA that was formed would be constant, approximately 0.35 μmol (1.45 mmol/g dry cells) in 10 min. By contrast, when ATP and Mg2+ were added into the assay mixture, reactivation would occur. Ten minutes later, the formed 3-HPA approached to 0.42 μmol, and then began to increase. When the reactivation occurred for 30 minutes, the 3-HPA was 0.80 μmol (3.20 mmol/g dry cells) and still showed the trend of rising.

Fig. 8 SDS-PAGE analysis of proteins in whole cells 1: protein markers (kD); 2: E. coli BL21 (pET-28gdrAB) absence of IPTG; 3: E. coli BL21 (pET-28gdrAB) after IPTG induction for 5 h; 4: E. coli BL21 (pET-32gldABC) absence of IPTG; 5: E. coli BL21 (pET-32gldABC) after IPTG induction for 5 h; 6: E. coli BL21 (pET-28gdrAB, pET-32gldABC) absence of IPTG; 7: E. coli BL21 (pET-28gdrAB, pET-32gldABC) after IPTG induction for 5 h. The positions of the products of all the genes are indicated by arrowheads.

Fig. 9 Characterization of glycerol dehydratase reactivase

3

Discussion

Glycerol dehydratase reactivase is a molecular chaperone of GDHt. The inactivated GDHt is rapidly reactivated by exchange of the modified coenzyme for intact coenzyme B12 in the presence of glycerol dehydratase reactivase, coenzyme B12, ATP, and Mg2+. Corinna and coworkers identified dhaF and dhaG in Citrobacter freundii as the genes responsible for the reactivation of the GDHt. The His6-tagged gene products of GDHt and reactivase were purified by Ni acid resin, and were then subjected to in vitro processed reactivation using purified components[15]. The study by Takamasa showed that a complex consisting of GdrA and GdrB was postulated as the reactivase because toluene-treated recombinant E. coli strains expressed the corresponding genes by compatible plasmids and reactivated the glycrol-inactivated GDHt[7]. In this study, the recombinant E. coli containing pET-32gldABC and pET28gdrAB, two incompatible plasmids, were selected under screening pressure by ampicillin and kanamycin. The activity of glycerol dehydratase reactivase was then identified by coexpression of these two incompatible plasmids in E. coli BL21 (DE3). It has been confirmed in this study that under screening pressure, the two incompatible plasmids can be used in coexpression of recombinant proteins in E. coli. In this study, gdrA and gdrB were amplified from the genomic DNA of K. pneumoniae, and the cloning vector pMD-gdrAB was constructed, which was identified by DNA sequence analysis. The fragment of gdrAB was subcloned into the expression vector to construct pET-28gdrAB, and then the recombinant E. coli containing pET-32gldABC and pET28gdrAB was selected from the medium under screening pressure by ampicillin and kanamycin. After incubation by IPTG, two incompatible plasmids coexpressed the GDHt and reactivase in E. coli. This was the first demonstration of such reactivation for GDHt using two coexpression products of incompatible plasmids. When the GDHt recovered its activity in the presence of ATP, Mg2+, coenzyme B12, and reactivase, quantity of the formed 3-HPA was about two times more than that of the medium in which reactivation had not occurred. When the reactivation occurred for thirty minutes, the formed 3-HPA still showed the trend of rising. In this study, it was confirmed that two incompatible plasmids could be used for coexpressing the recombinant proteins in E. coli under the screening pressure by two kinds of antibiotics. Moreover, it identified that the inactivated GDHt could be reactivated by glycerol dehydratase reactivase in presence of ATP, Mg2+, and coenzyme B12. Constructing the recombinant E. coli provides an opportunity for producing 3-HPA and plays the basic role in deeper research for reactivation. B

B

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adenosylcobalamin-dependent diol dehydratase and glycerol

REFERENCES

dehydratese. Archives of Microbiology, 2000, 174: 81–88. [1]

El-Ziney MG, van-den-Tempel T, Debevere J. Application of reuterin produced by Lactobacillus reuteri 12002 for meat

mechanism

adenosylcobalamin-dependent

factor

dehydratase.

for The

Structure, 2003, 11: 109–119. [11] Yang W, Zhang L, Lu ZG, et al. Coexpression of DNA

1,3-Propanediol

fragmentation factor subunits in E. coli by two incompatible

manufacture by epoxide hydrocarbonylation. US 4935554,

plasmids. Acta Biochimica et Biophysica Sinica, 2001, 33 (2):

Murphy

MA,

Smith

BL,

Aguilo

A.

238–242.

Xu ZH, Guo SZ, Wang PL. Preparation of 1,3-propanediol

[12] Fan LQ, Yuan QS, Wu XF. Coexpression of caiB and caiE with

based on hydration of acrolein with subsequent hydrogenation.

incompatible plasmids in E. coli. Chinese Journal of Pharmaceuticals, 2002, 33(3): 113–116. [13] Pang YQ, Jia HG, Fang RX. A simple and visible assay for cre

Tetsuo T. Radical catalysis of B12 enzymes: structure,

recombinase activity in Escherichia coli by using two

mechanism, inactivation, and reactivation of diol and glycerol

incompatible plasmids. Acta Microbiologica Sinica, 2005,

dehydratases. Cellular and Molecular Life Sciences, 2000, 57: 106–127.

45(1): 125–128. [14] Tetsuo T, Kazutoshi U, Saburo F, et al. Studies on the

Susumu H, Tetsuo T, Saburo F. In situ reactivation of

mechanism of the adenosylcobalamin-dependent dioldehy-

glycerol-inactivated

dratase reaction by the uses of analogs of the coenzyme. The

coenzyme

B12-dependent

enzymes,

glycerol dehydratase and diol dehydratase. Journal of Bacteriology, 1980, 143(3): 1458–1465.

[8]

reactivating

glycerol

dehydratase reactivase: a new type of molecular chaperone.

21–24.

[7]

a

metabolites of Lactobacillus reuteri on microorganisms and

Petroleum Processing and Petrochemicals, 2001, 32(12):

[6]

of

Journal of Biological Chemistry, 2001, 276(39): 36514–36519.

1990.

[5]

action

[10] Liao DI, Reiss L, Turner I, et al. Structure of glycerol

Chemical Society, 1999, 37: 117–125.

[4]

of

Chen CC, Chen JY, Lee SR. Growth inhibition of glycerol human cancer cell lines. Journal of Chinese Agricultural

[3]

Hideki K, Koichi M, Takamasa T, et al. Characterization and

decontamination and preservation. Journal of Food Protection, 1999, 62: 257–261. [2]

[9]

Journal of Biological Chemistry, 1977, 252(3): 963–970. [15] Corinna S, Susanne B, Gerhard G, et al. Identification and

Takamasa T, Hideki K, Michio Y, et al. Identification and

expression of the genes and purification and characterization of

expression of the genes encoding a reactivating factor for

the gene products involved in reactivation of coenzyme

adenosylcobalamin-dependent glycerol dehydratase. Journal of

B12-dependent glycerol dehydratase of Citrobacter freundii.

Bacteriology, 1999, 181(13): 4110–4113.

Journal of Biochemistry, 2001, 268: 2369–2378.

Takamasa T, Hideki K, Tetsuo T. Specificities of reactivating factors for

B