Molecular Cloning and Co-Expression of Phytoene Synthase Gene from Kocuria gwangalliensis in Escherichia coli

Abstract

A phytoene synthase gene, crtB, was isolated from Kocuria gwangalliensis. The crtB with 1,092 bp full-length has a coding sequence of 948 bp and encodes a 316-amino-acids protein. The deduced amino acid sequence showed a 70.9% identity with a putative phytoene synthase from K. rhizophila. An expression plasmid, pCcrtB, containing the crtB gene was constructed, and E. coli cells containing this plasmid produced the recombinant protein of approximately 34kDa , corresponding to the molecular mass of phytoene synthase. Biosynthesis of lycopene was confirmed when the plasmid pCcrtB was co-transformed into E. coli containing pRScrtEI carrying the crtE and crtI genes encoding lycopene biosynthetic pathway enzymes. The results obtained from this study will provide a base of knowledge about the phytoene synthase of K. gwangalliensis and can be applied to the production of carotenoids in a non-carotenoidproducing host.

Introduction

Carotenoids are natural pigments found in bacteria, fungi, algae, and plants. They are important compounds in isoprenoid metabolism. Depending on the structure, carotenoids can have various colors such as red, yellow, or orange and they play a critical role in the human body. Representative functions for carotenoids include protection from photo-oxidative damage, vitamin A precursors, anti-cancer effects, antioxidant effects, and immunity boosting. The carotenoids are widespread amongst bacteria and undoubtedly play an important role in protecting them from the damaging effects of light and, in an aerobic environment, the oxidative damage from activated forms of oxygen. Carotenoids may also play a role as light-gathering pigments in photosynthetic bacteria.

They have recently been studied for use in commercial applications such as cosmetics, food additives, and nutritional supplements [10,22]. To date, most carotenoids are known to have a C40 hydrocarbon backbone structure and 3-15 double bonds, and the absorption maxima are reported to be at 400-500 nm [3,10]. The microorganisms that are known to synthesize these carotenoid antioxidants include Agrobacterium aurantiacum [31], Paracoccus marcusii [11], P. carotinifaciens [29], Paracoccus sp. M BIC1143 [20], P. haeundaensis [15,16,26], Phaffia rhodozyma [19], and Haematococcus pluvialis [4].

The biosynthetic pathway for carotenoids include sequential condensation of C5 isopentenyl pyrophosphate (IPP), derived from isoprenoid biosynthesis, to eventually produce C40 astaxanthin, as shown in Fig. 1. The focus of this study was on phytoene synthase, which produces phytoene, the first product in carotenoid biosynthesis. Phytoene is a colorless carotenoid and is formed by the condensation of two geranylgeranyl pyrophosphate (GGPP) molecules, as observed in Fig. 1. This is the first step in the biosynthesis of the C40 family carotenoids. Phytoene is a precursor for various carotenoids such as β-carotene, lycopene, zeaxanthin, canthaxanthin, and astaxanthin; it normally does not accumulate in the organism and comprises the smallest part of the total carotenoids synthesized by microorganisms [24]. Phytoene synthase (CrtB), the enzyme involved in phytoene biosynthesis, uses Mn2+ as a cofactor and has the same structural function as other C30 carotenoid synthases such as dehydrosqualene synthase (CrtM) and squalene synthase [8]. The phytoene synthase gene (crtB) has been reported in P. haeundaensis [15], Kocuria rhizophila [28], Arthrobacter arilaitensis [12], Brachybacterium faecium [6], Micrococcus luteus [32], Sanguibacter keddieii [9], Microbacterium testaceum [5], Marine actinobacterium [18], Leifsonia xyli [7], and Corynebacterium glutamicum [14], to date.

Fig. 1.Summary of the lycopene biosynthetic pathway. Abbreviations used: FPP, farnesyl pyrophosphate; IPP, isopentenyl pyrophosphate (diphosphate); CrtE, GGPP synthase; GGPP, geranylgeranyl pyrophosphate; CrtB, phytoene synthase; CrtI, phytoene desaturase.

Kocuria gwangalliensis produces pink-orange pigments when cultured on laboratory media. Pigments produced by cold-loving microorganisms can be used instead of synthetic ones as alternative safe and natural colors in the food industry (ice-cream, candies, and other colored food products) and pharmaceuticals. Enzymatic oxidative cleavage of carotenoids is also found in bacteria and plants. We cloned the crtB gene, which encodes phytoene synthase involved in phytoene biosynthesis, from the genomic DNA of the marine bacterium K. gwangalliensis [25], which produces a pink-orange pigment. By co-expression of the crtE and crtI genes from P. haeundaensis, which we have previously isolated, and the crtB gene from newly isolated K. gwangalliensis, we induced and confirmed the biosynthesis of lycopene in Escherichia coli. The results of this study will lead to an understanding of carotenoid biosynthesis in K. gwangalliensis and provide the data for increasing the production of carotenoids in a non-carotenoid-producing host.

 

Materials and Methods

Bacteria Strains and Culture Condition

The microorganism K. gwangalliensis, which produced pink pigments, was isolated from Gwangalli Beach in Busan, Korea and cultured in PPES-II (Polypeptone 0.2 g, Bacto soytone 1.0 g, Proteose peptone 1.0 g, Bacto yeast extract 1.0 g, NaCl 3%, Ferric citrate 0.1%, DW 1 L) medium at 25℃. The E. coli XL1-blue [endA1 gyrA96 (nalR) thi-1 recA1 relA1 lac glnV44 F' [::Tn10 proAB+ lacIq Δ(lacZ) M15] hsdR17(rK- mK+) ] and BL21 (DE3) [F– ompT gal dcm lon hsdSB (rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])] strains were cultured in Luria-Bertani (LB; Tryptone 1%, Yeast extract 0.5%, NaCl 1%) medium at 37℃.

Construction of a Genomic DNA Library

Chromosomal DNA was prepared from cells of K. gwangalliensis grown in PPES-II. Late-exponential phase cells were harvested by centrifugation at 5,100 ×g for 10 min at room temperature and washed with 50 ml of TE buffer (10 mM Tris-HCl, pH 7.5, and 1 mM ethylenediamine tetraacetic acid). The cells were collected by centrifugation at 11,000 ×g for 10 min at room temperature, resuspended in 9 ml of TE buffer containing 5 mg/ml lysozyme and 100 μg/ml RNase A (Sigma Chemical Company, St. Louis, MO, USA), and then incubated at 37℃ for 1 h. After the addition of 0.5 ml from 10% (w/v) SDS and 0.1 ml of 10 mg/ml proteinase K, the suspension was incubated at 37℃ with shaking for several hours to complete the lysis. The lysate together with 1.8 ml of 5 M NaCl and 1.5 ml of 10% (w/v) acetyltrimenthylammonium bromide (CTAB) in 0.7 M NaCl was incubated at 65℃ for 20 min. An equal volume of chloroform/isoamyl alcohol (24:1 (v/v)) was added, and the mixture was gently mixed by inverting the tube at room temperature for 10 min. The phases were separated by centrifugation at 11,000 ×g for 20 min at room temperature. After two extractions with chloroform/isoamyl alcohol, the aqueous phase was extracted once with an equal volume of phenol saturated with 1 M Tris-HCl (pH 7.5) and again with chloroform/isoamyl alcohol. A 2-fold volume of cold ethanol (-20℃) was added and the precipitated DNA was collected using a glass rod and was rinsed with 70% (v/v) ethanol. The ethanol was evaporated under vacuum, and the DNA was suspended in 3 ml of TE buffer. The DNA concentration was determined by measuring the absorbance of a 20-fold dilution at 260 and 280 nm. The purified chromosomal DNA of K. gwangalliensis was partially digested by incubation at 37℃ for 1 h with 5 kU Sau3AI/g genomic DNA. A genomic DNA library of K. gwangalliensis was constructed using a ZAP Express BamHI/Gigapack III cloning kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. The resulting library contained approximately 1 × 105 clones. The library was amplified to 3 × 109 clones per milliliter.

Cloning and Sequencing of Phytoene Synthase Gene

To clone the phytoene synthase gene (crtB) from K. gwangalliensis, the conserved sequence of phytoene synthase from phylogenetically related bacteria was identified from the NCBI (National Center for Biotechnology Information, USA) conserved domain site, and a degenerate primer was designed using the identified sequence (Table 1). The sequence was amplified using the degenerate primer by polymerase chain reaction (PCR). To clone the open reading frame of crtB, a DNA Walking SpeedUp Premix Kit (Seegene, Korea) was used. The amplified DNA was inserted into pGEM-T vector (Promega, USA) and cloned. The complete DNA sequence of crtB was analyzed and registered in GenBank (Accession No. JN582050).

Table 1.Primers used in this study.

Analysis of DNA Sequence of K. gwangalliensis Phytoene Synthase Gene (crtB)

To analyze the sequence of K. gwangalliensis crtB DNA, the BLAST algorithm from the GenBank database was used. The homology of known bacteria was investigated by analyzing the amino acid sequence of phytoene desaturase. The multiple sequence alignment of amino a cids w as a naly zed by u sing the C lustalW program. To analyze the phylogenetic tree, the Neighbor (MEGA, ver. 4.0) program that employs the neighbor-joining method was used [23].

Construction of Plasmid for Lycopene Synthesis in E. coli

To assess whether KgCrtB can be expressed in E. coli, we cloned the KgCrtB gene into the NdeI/HindIII site of the pColdI vector (TaKaRa, Japan). We amplified the genes by using primers (Table 1) that had an NdeI at the N-terminus and a HindIII site at the C-terminus, digested the product by using restriction enzymes, subcloned it into the pColdI vector, and called this plasmid pCcrtB. In addition, to synthesize lycopene in E. coli, we cloned the P. haeundaensis crtE and crtI genes in the MCS1 and MCS2 sites, respectively, of the pRSFDuet-1(Novagen, USA) co-expression vector. To clone the crtE (GGPP synthase) gene into MCS1, we designed an EcoRI restriction site and a NotI restriction site at the N- and C- terminal, respectively, and to clone the crtI (phytoene desaturase) into MCS2, we designed an NdeI restriction site and XhoI restriction site at the N- and C- terminal, respectively. The completed recombinant plasmid was named pRSCrtEI. Following this, the selection of transformed E. coli was conducted by ampicillin resistance. Transformed E. coli was cultured in LB medium containing 50 μg/ml of ampicillin at 37℃ until OD600 reached 0.5. Following this, 0.1 mM IPTG was added at 16℃ to induce the overexpression of CrtB. To analyze the expression level, 12% SDS-PAGE was conducted. The proteins were electrotransferred from the SDS-PAGE gel to a nitrocellulose membrane, probed with goat antiserum against 6-His tag, and incubated with alkaline phosphatase coupled to goat antibody against goat IgG. The nitrocellulose membrane was developed using BCIP/NBT (Sigma-Aldrich, USA) [27]. Separately, the crtE and crtI genes from P. haeundaensis were inserted into the pRSFDuet-1 vector (Novagen, USA) to synthesize lycopene in E. coli BL21 (DE3). The resulting pRScrtEI plasmid was transformed into E. coli BL21 (DE3) followed by a kanamycin selection process. To synthesize lycopene in E. coli, the two plasmids constructed (pCcrtB and pRSCrtEI) were co-transformed and co-expressed in BL21 (DE3) at 37℃ for 24 h culture.

Extraction and Analysis of Lycopene in E. coli

Biosynthesized lycopene from E. coli was analyzed using HPLC. To extract the pigment from transformed bacteria, the cells were homogenized using 90% acetone for 3 min followed by a 2 min centrifugation at 13,000 rpm. The supernatant was collected and filtered (0.22 μm nylon filters). Then, 20 μl of this sample was injected into an HPLC (Bio-Rad, USA) Automated Biologic HR System (#750-0047). The Nova-Pak HR 6 U C18 column was used at 40℃. The solvent was composed of 0.1 M Tris-HCl at pH 8.0; acetonitrile, methanol, and ethyl acetate were used for extracting the pigment. The solvent compositions for HPLC separation are as follows: 14% 0.1 M Tris-HCl (pH 8.0), 84% acetonitrile, 2% methanol (0–15 min); 68% methanol and 32% ethyl acetate (15–20 min). After the HPLC analysis time, a 10-min post-run was performed; the solvent compositions were identical to that of the starting solvent. The flow rate for the HPLC column was 1 ml/min. The separated carotenoids were recorded at absorption maxima of 280 and 470 nm by using a photodiode array detector. The readings were compared and analyzed with the standards, GGPP, lycopene, and phytoene (Sigma-Aldrich, USA).

 

Results and Discussion

Cloning of K. gwangalliensis Phytoene Synthase Gene

We constructed a genomic DNA library from the isolated K. gwangalliensis genomic DNA, and the genomic DNA library was used as templates for PCR. The primers used to clone the phytoene synthase gene (crtB) were degenerate primers designed by analyzing the sequences of various characterized phytoene synthases from other bacteria and by using regions that are conserved (Table 1). We were able to amplify a fragment of about 700 bp by using PCR with these primers. On the basis of the sequence of this fragment, a new set of target-specific primers (Table 1) was used for DNA walking to eventually amplify a 950 bp PCR product that was then TA-cloned and sequenced to determine the genomic sequence of K. gwangalliensis phytoene synthase (KgcrtB) (Fig. 2). The ORF (open reading frame) of the cloned KgcrtB was 948 bp, which encoded 316 amino acids, and the stop codon (TGA) was present at 946–948 bp. To identify the start codon, we searched for the gram-positive ribosome-binding site determined by the Shine-Dalgarno sequence and Morran et al. [21]. AGGA, a common prokaryotic sequence, was present 5 bp upstream of the start codon. In addition, the predicted molecular mass of the phytoene synthase encoded by the KgcrtB gene was 34.0 kDa and the predicted isoelectric point was slightly acidic at 6.21.

Fig. 2.Nucleotide and deduced amino acid sequences of the Kocuria gwangalliensis CrtB. The nucleotide sequence is numbered to the left and the amino acid to the right. The bold type indicates the initiator codon. The asterisk and bold type indicate the stop codon. The putative Shine-Dalgano sequences for the ribosome-binding site are in italic and bold type. The putative helical and beta-sheet are in dash-line and underline, respectively. The nucleotide sequence has the accession number JN582050 in the GenBank database.

Amino Acid Sequence Analysis of Phytoene Synthase (KgCrtB)

With respect to the amino acid sequence of the phytoene synthase encoded by KgcrtB, we used the SABLE website (http://sable.cchmc.org) to predict the secondary structure of the protein and Prediction of Transmembrane Regions and Orientation (TMpred) algorithm (http://www.ch.embnet.org) to predict the transmembrane domains. The secondary structure of the KgCrtB protein was predicted to have 2 β-sheets, 13 α-helices, and 16 random coils (Fig. 2). The predicted transmembrane domains exist between amino acids 62-81 and 144-160, with the N-terminal being on the inside.

To determine the conserved regions and phylogenetic relationships, we performed a multiple alignment by using previously reported bacterial phytoene synthase genes from GenBank (Fig. 3). In addition, to assess the phylogenetic relationships, we built a phylogenetic tree by using multiple alignment data (Fig. 4). Phytoene synthase is the first step in synthesizing C40 family carotenoids, and it is an important enzyme in determining carotenoid synthesis [13]. From the multiple alignment analysis, we were able to identify four conserved regions, and these are shown in Fig. 3 as Domains I–IV. The AFQ(XX)NXXRDL motif in Domain IV of phytoene synthase provides a site for Mg2+/Mn2+ binding to the substrate and is known to be a very hydrophobic region [1]. When catalytic activity was tested by enzyme inactivation, the Arg residue is thought to play an important role in the phytoene synthase [2]. This Arg residue can bind to the diphosphate anion and is proximal to an Asp-rich motif (Fig. 3).

Fig. 3.Multiple alignment of deduced amino acid sequences of KgCrtB and other bacteria. The amino acid sequences were obtained from GenBank: K. rhizophila (NC010617), Micrococcus luteus (YP002958145), Arthrobacter arilaitensis (YP003916549), Sanguibacter keddieii (ACZ21218), Brachybacterium faecium (YP003153907), C. glutamicum (YP224917), Leifsonia xyli (YP062472), Marine actinobacterium (ZP01129025), Microbacterium testaceum (BAJ76096), Paracoccus haeundensis (AY957386), Kocuria gwangalliensis (JN582050). Domains I-IV are conserved regions of phytoene synthase (KgCrtB).

Fig. 4.A molecular phylogenetic tree of CrtB based on the neighbor-joining method. Numbers at nodes indicate levels of bootstrap support based on 1,000 replicated datasets. Bar, 0.1 substitution per amino acid position.

To determine the phylogenetic relationship of KgCrtB, we performed multiple alignment analysis by using bacterial phytoene synthases. The homology to KgCrtB was as follows: Kocuria rhizophila (71%, Accession No. NC010617), Arthrobacter arilaitensis (64%, Accession No. YP003916549), Microbacterium testaceum (51%, Accession No. BAJ76096), Sanguibacter keddieii (50%, Accession No. ACZ21218), Leifsonia xyli (48%, Accession No. YP062472), Brachybacterium faecium (48%, Accession No. YP003153907), P. haeundaensis (47%, Accession No. AY957386), C. glutamicum (47%, Accession No. YP224917), Marine actinobacterium (41%, Accession No. ZP01129025), and Micrococcus luteus (19%, Accession No. YP002958145). Of these, the highest homology was observed with the phytoene synthase of K. rhizophila, which is in the same genus, and the lowest homology was observed with the phytoene synthase of M. luteus.

Expression of Phytoene Synthase Gene (crtB) in E. coli

To express crtB in E. coli, we transformed E. coli BL21 (DE3) with the pCcrtB plasmid. The selected strain was grown in LB medium containing 50 μg/ml of ampicillin at 37℃ until the OD600 reached 0.5, and the recombinant DNA was induced to overexpress with IPTG at a final concentration of 0.1 mM at 16℃. The expression pattern of the crtB gene is shown in Fig. 5A; the molecular mass of the expressed CrtB protein turned out to be approximately 34 kDa (predicted from amino acid sequence analysis; Fig. 5A). The optimal induction of a recombinant CrtB protein was achieved at 17 h after induction. The recombinant CrtB protein tagged with His-residues was confirmed by western blot analysis and is shown in Fig. 5B.

Fig. 5.Analysis of the expressed proteins using SDS-PAGE and western blotting. (A) The expressed proteins were analyzed using 12% SDS-PAGE. Lane M, standard protein molecular weight markers; lane C, proteins from uninduced cell extracts (control); lanes 1-6, proteins from induced cell extracts 0, 1, 3, 5, 7, and 17 h after IPTG induction, respectively. (B) Western blot analysis of expressed proteins. Lanes M-6, proteins used in the same order as loaded. A protein that is estimated to CrtB it was indicated by the arrow.

Extraction and Analysis of Carotenoids Synthesized in E. coli through Co-Expression

To determine the activity of KgCrtB expressed in E. coli BL21 (DE3), we assessed the biosynthesis of lycopene in E. coli. Normally, E. coli synthesizes FPP and IPP through the non-mevalonate pathway, but because it does not have crtE, crtB, and crtI genes, it cannot synthesize lycopene. Using this characteristic, we transformed E. coli BL21 (DE3) with the two recombinant plasmids mentioned above. The lycopene produced in the transformed strain was extracted and analyzed by HPLC. The absorption maxima of the carotenoids differ depending on the double bond, resonance structure, and carbon chain length. The absorption maxima of phytoene and GGPP are 280 nm and that of lycopene is 470 nm [30]. Fig. 6A shows the results of the standards GGPP, phytoene, and lycopene. Lycopene was isolated at 470 nm and retention time of 22 min (peak 3), GGPP was isolated at 280 nm and retention time of 13 min (peak 1), and phytoene was isolated at 280 nm and retention time of 27 min (peak 2). Fig. 6B shows the results of the carotenoid produced by the co-expression of pCcrtB and pRSCrtEI recombinant DNA and analyzed by using HPLC. Compared with the standards, we observed that lycopene was produced from the material isolated at 470 nm and retention time of 22 min. At 280 nm and retention time of 8 (peak 4), 13 (peak 1), and 27 min (peak 2), we confirmed the production of GGPP and phytoene, respectively. To enhance the production of lycopene, we focused on improving the conversion of GGPP to lycopene. Overexpression of KgCrtB in the E. coli strain was tested. Whereas pET-44(a)-CrtEBI [17] overexpression showed an effect on lycopene production (1.1 mg/g DCW), it could be shown that lycopene accumulation was increased 3-fold when pCcrtB and RScrtEI were co-overexpressed (3.4 mg/g DCW). Taken together, we concluded that KgCrtB did not lose its activity and functioned normally when transformed into an E. coli strain.

Fig. 6.HPLC analysis of carotenoids from E. coli BL21 (DE3). Detection at 280 nm and 470 nm. (A) GGPP (pick 1), phytoene (pick 2), and lycopene (pick 3) standard. (B) E. coli containing pCcrtB and pRScrtEI plasmids. Peak 1, GGPP. Peak 2, phytoene. Peak 3, lycopene. Peak 4, putative FPP or IPP.

In this study, we have isolated the crtB gene that encodes the enzyme required for the first step of the carotenoid biosynthesis pathway in K. gwangalliensis and analyzed the secondary structures of the KgCrtB protein to determine the structural characteristics of this enzyme. In addition, to test the activity of the enzyme, we transformed E. coli BL21 (DE3) with this gene to produce phytoene and lycopene and analyzed the products by using HPLC. These results can provide the foundational data for identifying new carotenoid biosynthesis enzymes and producing various carotenoids in a non-carotenoid-producing host.

The GenBank accession number for the CrtB nucleotide sequence reported here is JN582050.