Cloning and Characterization of a Novel Glutathione Transferase Gene from Penicillium chrysogenum

CHINESE JOURNAL OF BIOTECHNOLOGY Volume 23, Issue 4, July 2007 Online English edition of the Chinese language journal

Cite this article as: Chin J Biotech, 2007, 23(4), 618–622.

RESEARCH PAPER

Cloning and Characterization of a Novel Glutathione Transferase Gene from Penicillium chrysogenum ZHANG Yuan1,2, WANG Fu-Qiang1,2, ZHENG Gui-Zhen2, DAI Meng2, LIU Jing2, ZHAO Ying2, REN Zhi-Hong2, JIA Qian1,2*, ZHAO Bao-Hua1* 1

College of Life Science, Hebei Normal University, Shijiazhuang 050016, China

2

New Drug R&D Center, North China Pharmaceutical Co., Shijiazhuang 050015, China

Abstract:

Glutathione transferases (GSTs) are a family of multifunctional proteins that mainly catalyze the conjugation of

intracellular glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds. GSTs play important roles in stress tolerance and in the detoxification metabolism in organisms. A novel GST gene, PcgstB, was cloned from penicillin producing fungus Penicillium chrysogenum using RT-PCR. The open reading frame (ORF) of PcgstB was 651bp and encoded a peptide of 216 residues. The deduced amino acids sequence had conserved GST domain and showed 65% identity to the characterized Aspergillus fumigutus gstB. The entire ORF of PcgstB was inserted into vector pTrc99A and transformed into Escherichia coli DH5α. Recombinant PcGstB was overexpressed and its GST activity toward substrate 1-chloro-2,4-dinitrobenzene (CDNB) was validated. Key Words:

glutathione transferase; Penicillium chrysogenum; recombinant expression; enzyme assay

Glutathione transferases (GSTs, EC 2.5.1.18) are a family of multifunctional proteins that exist in plants, animals, and microbes. These are divided into three major types: cytosolic GSTs, mitochondrial GSTs, and microsomal GSTs[1]. GSTs catalyze the conjugation of glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds, which facilitates to form stable and resolvable compounds and to be pumped out of body for detoxification[2]. Furthermore, GSTs are also involved in compound transformation, cellular signal control, and so on. At present, researches on GSTs mainly focus on mammals, plants, and insects. In animals, GSTs can protect their bodies from attacks of toxic substances; in plants, GSTs play an important role in the detoxification of pesticide and herbicide. The tertiary structure of most cytosolic soluble GSTs is globular dimmer. The molecular weight of the subunits ranges from 23 kD to 29 kD. Each subunit consists of two distinct

domains: the N-terminal domain evolves to bind GSH, and the C-terminal domain appears to combine with electrophilic compounds, which may be responsible for different substrate characteristics. According to the amino acid sequence, the substrate specificity, the sensitivity to inhibitors, and the immunological characteristics, GST can be sorted into several classes. Soluble GSTs in mammals involve 8 types, α, μ, π, σ, θ, ζ, κ, and ω; besides these, there are φ and τ GSTs only in plants, δ and ε in insects, and β GST in bacteria[3]. Up to date, the studies on GSTs in fungi are relatively rare and most characterized fungal GSTs are different from these current classes[4]. The filamentous fungi Penicillium chrysogenum is an important strain that is used for industrial producing of β-lactam antibiotic-penicillin, which has significant business value. Several studies have shown that the metabolism of GSH can be closely relevant to penicillin biosynthesis in P.

Received: November 23, 2006; Accepted: December 21, 2006. * Corresponding authors. ZHAO Bao-Hua: Tel: +86-311-86268434; Fax: +86-311-86268313; E-mail: [email protected] JIA Qian: Tel: +86-311-86057649; Fax: +86-311-6676507; E-mail: [email protected] This work was supported by a grant from the National Key Sciences and Technologies R&D Program of China (2002BA711A16). Copyright © 2007, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.

ZHANG Yuan et al. / Chinese Journal of Biotechnology, 2007, 23(4): 618–622

chrysogenum. On one hand, GSH (γ-L-glutamylL-cysteinyl-glycine) is structurally similar to δ-(L-α-Aminoadipyl)-L-cysteinvl-D-valine (LLD-ACV), which is a crucial intermediate of the penicillin biosynthesis[5]. High concentration of GSH will restrain the activity of key enzymes ACV synthetase in this procedure, and influence the penicillium production[6]. On the other hand, GSH may take part in the activation or degradation of phenylacetate acid, the side-chain precursor of penicillin G[7–10]. However, the real function of GSH in penicillin biosynthesis remains obscure. Identification of genes that involve in the GSH metabolism in P. chrysogenum may help us to reveal the relationship of GSH metabolism and penicillin biosynthesis genetically. In this article, we report the molecular cloning and functional identification of a novel GST gene from P. chrysogenum.

1

Materials and methods

1.1 Materials 1.1.1 Strains: P. chrysogenum Wis54-1255 (ATCC28089) was used in this study and Escherichia coli DH5α (Invitrogen) was used for nucleic acids manipulations and prokaryotic expression. 1.1.2 Vectors and reagents: The prokaryotic expression vector pTrc99A was purchased from Phamacia, and the cloning vector pGEM-T was purchased from Promega. Restriction endonucleases, T4 DNA ligase, Taq DNA polynerase, dNTP, and Random primer（Random Hexamers） were also products of Promega. The DNA Ligation Kit Ver2.0 was purchased from TaKaRa. The EZ-10 spin column DNA Gel Extraction kit was produced by BIO BASIC. SuperScriptTM Ⅱ RNase H was purchased from Invitrogen. 1-Chloro-2, 4-dinitrobenzene (CDNB) was purchased from Sigma. The LMW Calibration kit for SDS Electrophoresis was obtained from Amersham Biosciences. 1.2 Methods 1.2.1 Isolation of RNA from P. chrysogenum: The total RNA of P. chrysogenum (Wis54–1 255) was isolated by Trizol (Invitrogen) according to the user’s manual. Genomic DNA contamination was eliminated by RQ1 RNase-free DNase (Promega). Two microgrammes total RNA were utilized to construct the first strand cDNA using the SuperScriptTMⅡRNase H (Invitrogen) and Random Hexamers. 1.2.2 Cloning of P. chrysogenum gst B: Based on our previous study, a pair of primers was designed to amplify the entire CDS of P. chrysogenum gstB by RT-PCR using Wis 54–1 255 cDNA as the template; the forward primer P1: 5′-CCATGGAATCTCTCAAACCTATCA-3′ and the reverse primer P2: 5′-GTCGACTTAGCGCCGCGCACAAGC-3′. Nco I and Sal I sites were introduced to the 5′ and 3′ ends, respectively. The PCR conditions were as follows: predenaturation at 94˚C for 5 min; 35 cycles at 94˚C denaturing for 30 s, 55˚C annealing for 1 min, 72˚C extension

for 1 min, and then a final step of 72˚C for 10 min. The total volume was 50 µL. The amplification fragment was ligated into the pGEM-T vector and sequenced. 1.2.3 Phylogenetic analysis: The multiple alignment of the deduced P. chrysogenum GstB amino acid sequence with the GSTs sequence of known fungal GSTs from GenBank was made by ClustalW. The phylogenetic tree was constructed using the neighbor-joining (NJ) method and the MEGA program. The data were analyzed using the p-distance model with 1 000 bootstrap tests. 1.2.4 Heterologous expression of PcgstB in E. coli: The ORF of the PcgstB gene was excised from pGEMT-gstB by Nco I and Sal I and inserted into the expression vector pTrc99A, which was digested by the same restriction endonucleases, to generate plasmid pTrc-gstB. E. coli DH5α transformed with pTrc-gstB was used to overexpress the recombinant PcGstB according to the standard manual. After culture in Luria-Bertani (LB) broth containing ampicillin (100 μg/mL) overnight at 37˚C, DH5α/pTrc-gstB cells were inoculated into the same liquid medium at one percentage concentration and grown at 37˚C until A600 reached 0.4 to 0.6. The expression of the recombinant GstB protein was induced with 0.5 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) for 4 h at 37˚C and analyzed by 12% SDS-PAGE. Simultaneously, E. coli DH5α containing plasmid pTrc99A was used as a control. To determine the form of recombinant PcgstB in E. coli cells, bacteria of DH5α/pTrc-gstB were resuspended in 10 volumes of buffer A (50 mmol/L Tris-HCl, 0.5 mmol/L EDTA, 50 mmol/L NaCl, 5% (W/V) glycerol, pH 7.5) and sonicated on ice-water bath. 1.2.5 GST activity assay: The GST activity was measured spectrophotometrically using CDNB and GSH as substracts[11]. One gramme of DH5α/pTrc-gstB or DH5α/pTrc99A wet cells was dissolved in 10 mL of buffer A and sonicated. The cell debris was removed by centrifugation at 13 000 g for 10 min at 4˚C. One milliliter of the supernatant was used for enzyme assay. The total enzymatic reaction system was 3 mL containing 0.1 mmol/L phosphates (pH 6.5), 1.0 mmol/L CDNB, and 1.0 mmol/L GSH. The reactions were incubated at 30˚C for 30 min. The GST activity was determined by measuring the increase in A340. A complete assay mixture without the supernatant was used as a control.

2

Results

2.1 Cloning of gstB cDNA from P. chrysogenum In our former study, a GST gene, PcgstA, was cloned for the first time from P. chrysogenum by degenerating primer PCR and screening of genomic library[12]. We analyzed the else clones that were gained from hybridization, and found an open-reading frame of 651 bp, which encoded a protein of 216 amino acids residues. The deduced amino acid had conserved

ZHANG Yuan et al. / Chinese Journal of Biotechnology, 2007, 23(4): 618–622

GST regions (Fig. 1) and showed about 65% sequence identity with Aspergillus fumigatus GstB (GenBank accession No. AAX07318). To obtain the cDNA of the gene, a pair of PCR primers (P1/P2) was designed according to the genomic sequence and used for PCR with cDNA template from P. chrysogenum Wis54–1255. A 0.65 kp fragment was amplified (Fig. 2) and subcloned into vector pGEM-T. The sequencing result indicated that the sequence of the PCR product was just consistent with our anticipation. The gene was named PcgstB and deposited in GenBank under accession number EF123038.

the recombinant PcGstB protein was mainly present in cell lysate supernatant in soluble form.

Fig. 1 Conserved domain analysis of the deduced amino acids

The full-length PcgstB gene was amplified by PCR using P.

sequence of PcGstB

chrysogenum cDNA as a template. Line 1: PCR products of PcgstB

The result of BLASTp displayed that the deduced amino acid

gene; line 2: DNA Marker DL2000 with the size in bp.

Fig. 2 RT-PCR product of the PcgstB gene

sequence of PcGstB had two GST-like conserved regions: the N-terminal domain and the C-terminal domain.

GST can catalyze the conjugation of CDNB with GSH and the outcome has characteristic absorption at 340 nm, which can be

2.2 Sequence analysis of PcgstB We compared PcgstB with other fungal GSTs characterized to date. Phylogenetic analysis of the protein sequence (Fig. 3) showed that PcGstB are most closely related to the GstB and GstC (GenBank accession No. AAX07318 and AAX07319) from A. fumigates. The sequence identities were 65% and 52%, respectively, while Schizosaccharomyces pombe GstI (Q9Y7Q2) and GstI (O59827) also had significant relatedness to PcGstB (43% and 44% sequence identity, respectively). Just like PcgstB, none of the above four GST genes have introns. The other four filamentous fungi GSTs genes, BfGst1 (Botryotinia fuckeliana, AAG43132), AfGstA (A. fumigatus, AAX07321), AnGstA(Aspergillus nidulan, AAM48104), and PcGstA (P. chrysogenum, ABE73180), are all interrupted by two introns. Their protein sequence identities with PcGstB are 43%, 44%, 43%, and 37%, respectively, whereas the GSTs from Saccharomyces cerevisiae[13], Issatchenkia orientalis[14], and Cunninghamella elagans[15,16], are less related to PcGstB. 2.3 Expression and activity analysis of recombinant PcGstB To identify the function of protein encoded by PcgstB, we constructed the expression vector pTrc-gstB by ligating the entire ORF of PcgstB into vector pTrc99A under the control of the strong trc promoter (Fig. 4). pTrc-gstB was transformed into E. coli DH5α and the expression was induced by IPTG. DH5α transformed with empty vector pTrc99A was used as a control. SDS-PAGE analysis (Fig. 5) showed a clear band corresponding to approximately 28 kD appearing in induced DH5α/ pTrc-gstB cells. After sonication and centrifugation,

determined spectrophotometrically. According to the method of Habig et al[11], the cell lysate supernatant of DH5α/pTrc-gstB and DH5α/pTrc99A that induced by the addition of IPTG were assayed GST activity, respectively. Clearly, the GST activity was determined from DH5α/pTrc-gstB supernatant (Table 1). This result indicated that PcgstB encoded a functional GST assuredly.

3

Discussion

The industrialization and clinical application of penicillin can be called a splendent example of biotechnology[17]. In the past 60 years, several efforts have been made to improve the productivity of the penicillin industrial strain, P. chrysogenum. A complete breeding technology based on random mutation and subsequent screening has been built up and the yield has been enhanced about a thousand times. The development of molecule biology and genetic engineering and the cloning of the key genes in penicillin biosynthesis provide a more direct and efficient method for strain improvements. Phenylacetate acid (PAA), the side-chain precursor of penicillin G production, takes a large proportion in industrial fermentation cost. Improving the utility of phenylacetate acid is a major objective in strain improvements[18]. GSH metabolism in P. chrysogenum can be closely relevant to penicillin biosynthesis[5]; Ferrero et al[10] proposed a putative activating way of PAA relating to GST, but there is no evidence at molecular level. We cloned a GST encoding gene, PcgstA from P. chrysogenum for the first time. The expression of PcgstA was evidently down-regulated in the presence of

ZHANG Yuan et al. / Chinese Journal of Biotechnology, 2007, 23(4): 618–622

PAA, which indicated that it is likely involved in the

phenylacetate acid metabolism in P. chrysogenum.

Fig. 3 Phylogenetic analysis of th e characterized fungal GSTs The sequence of P. chrysogenum PcGstB protein was aligned with 16 known fungal GSTs from the GenBank by ClustalW. A neighbor-joined tree was created by the MEGA software with bootstrapping of 1 000.

Fig. 4 Identification of the recombinant plasmid pTrc-gstB Line 1: DL2, 000 DNA marker with the size in bp; line 2: thhe expression vector of pTrc-gstB digested by Nco I and Sal I. Two DNA

Fig. 5 SDS-PAGE analysis of the expression of recombinant protein

fragments, 3.0 kb pTrc99A vector and 0.65 kb inserted PcgstB ORF,

in E. coli

can be seen; line 3: λ-Hind Ⅲ DNA marker with size in bp.

The gel was stained with Coomassie brilliant blue. Line 1: the total proteins of DH5α/pTrc99A without induction; line 2: the total proteins of DH5α/pTrc99A induced by IPTG; line 3: the total proteins

Table 1 Analysis of recombinant PcGstB Sample

ΔA340/min

supernatant of DH5α/pTrc99A

0.0006

supernatant of DH5α/pTrc-gstB

0.0253

The GST activity was assayed with CDNB. The specific activity reported was an average of there replicates and was adjusted with blank reaction.

of DH5α/pTrc-gstB without induction; line 4: the total proteins of DH5α/pTrc-gstB induced by IPTG; line 5: low molecular weight protein markers with size in kD.

In this study, a novel GST gene, PcgstB, was cloned from P. chrysogenum, and overexpressed in E.coli. Enzymatic assay confirmed that the recombined PcGstB exhibited GST activity with CDNB as a substrate. PcGstB showed 37% sequence identity with P. chrysogenum PcGstA at the amino acid level, and 65% identity with A. fumigatus GstB. Phylogenetic analysis demonstrated the relationship between PcGstB,

ZHANG Yuan et al. / Chinese Journal of Biotechnology, 2007, 23(4): 618–622

PcGstA, and other fungal GSTs (Fig. 3). The expression analysis of the PcgstB gene and the cloning of other P. chrysogenum GST genes will help us to elucidate the relationship between GSTs, GSH, and the phenylacetate acid metabolism and their function in penicillin biosynthesis. Up to date, the study of GSTs in fungi, especially in filamentous fungi, is relatively less. The limited characterized fungal GSTs have large difference from that of other species. With the development of molecule biology and microbe genome researches, the identification of more and more fungal GST genes will enrich our knowledge of GST evolution.

[8]

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