molecular cloning and functional characterization of a Δ6-fatty acid desaturase gene from rhizopus...

Journal of Basic Microbiology 2012, 52, 1–5 1

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Molecular cloning and functional characterization of a ∆6-fatty acid desaturase gene from Rhizopus oryzae

Yu Zhu1 and Bi-Bo Zhang2

1 Department of Immunology, Chong Qing Medical University, Chongqing 400016, People’s Republic of China

2 College of Life Sciences, Chongqing University of Arts and Sciences, Chongqing 402168, People’s Republic of China

The objective was to screen for and isolate a novel enzyme with the specific activity of a Δ6-fatty acid desaturase from Rhizopus oryzae. In this study, R. oryzae was identified as a novel fungal species that produces large amounts of γ-linolenic acid. A full-length cDNA, designated here as RoD6D, with high homology to fungal Δ6-fatty acid desaturase genes was isolated from R. oryzae by using the rapid amplification of cDNA ends method. It had an open reading frame of 1176 bp encoding a deduced polypeptide of 391 amino acids. Bioinformatics analysis characterized the putative RoD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, a hydropathy profile, and a cytochrome b5-like domain in the N terminus. When the coding sequence was expressed in the Saccharomyces cerevisiae strain INVScl, the encoded product of RoD6D exhibited Δ6-fatty acid desaturase activity that led to the accumulation of γ-linolenic acid. The corresponding genomic sequence of RoD6D was 1565 bp in length, with five introns. This is the first report on the characterization and gene cloning of a Δ6-fatty acid desaturase of R. oryzae from Douchi.

Keywords: γ-Linolenic acid / ∆6-Fatty acid desaturase gene / Cloning / Rhizopus oryzae

Received: April 21, 2012; accepted: May 14, 2012

DOI 10.1002/jobm.201200189

Introduction*

Polyunsaturated fatty acids (PUFA) play important roles as structural components of membrane lipids and stor-age lipids in eukaryotic cells [1, 2]. The PUFA confer the biological properties of biomembranes, particularly the membrane fluidity and permeability, and also serve as signaling molecules in response to environmental stresses such as low temperature, salt stress, and pathogenic intrusion into cells [3]. The most significant sources of γ-linolenic acid (GLA) are plant seeds and microbial oils. The unsaturated fatty acids are formed by desaturases, which catalyze the introduction of dou-ble bonds into preformed acyl chains by the removal of hydrogen atoms and concomitant oxidation [4]. With the desaturase genes isolated from various organisms, the enzymatic reactions and corresponding metabolic

Correspondence: Yu Zhu, Department of Immunology, Chong Qing Medical University, Chongqing 400016, People’s Republic of China E-mail: [email protected] Phone: +86-23-86023165

pathways are well elucidated. Among them, Δ6-desaturase catalyzes the conversion of linoleic acid (LA, 18:2, Δ9, 12) and α-linolenic acid (18:3, Δ9, 12, 15) to GLA (18:3, Δ6, 9, 12) and stearidonic acid (18:4, Δ6, 9, 12, 15), respectively. Subsequently, the resultant fatty acids can be introduced into the biosynthesis of long-chain PUFA (LC-PUFA) through an alternating series of desaturation and elongation reactions. The Δ6-fatty acid desaturase gene has been previously cloned and charac-terized from several fungi, such as Mortierella alpine and R. nigricans [5]. Rhizopus oryzae phylogenetically belongs to the Mu-corales. In our previous work, we have isolated the R. oryzae strain DR3 from the fermentation of Douchi. Using GC analysis, we discovered that this species also synthesizes PUFA, and especially GLA. Identifying the genes associated with the synthesis of GLA will con-tribute to the characterization of the desaturase genes in R. oryzae, establishing a simple model for studying the metabolic pathways of GLA in eukaryotes and pro-viding the primary basis for the future application of

2 Yu Zhu and Bi-Bo Zhang Journal of Basic Microbiology 2012, 52, 1–5


genetic engineering to GLA production. In this work, we report the cloning and expression of the Δ6-de-saturase from the R. oryzae strain DR3.

Materials and methods

Chemicals GLA methyl ester and LA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bacto-yeast extract, 2% bacto-peptone, and other important medium compo-nents were purchased from Oxoid Ltd. (Hampshire, England). Organic solvents and other chemicals of analytical grade were from Sangon Biotech, Shanghai, China. The reagents for analysis were of GC-analytical grade. PCR amplification was performed using a ther-mal cycler from Eppendorf.

Organisms and growth conditions The R. oryzae DR3 strain was grown at 26 °C for 5 d in liquid-medium potato dextrose agar (PDA) with the pH adjusted to 6.8. The Saccharomyces cerevisiae strain INVScl was used as recipient in transformation experi-ments and was grown at 30 °C in complex medium (yeast extract peptone dextrose, YPD) containing 1% bacto-yeast extract, 2% bacto-peptone, and 2% glucose.

Cloning of a full-length cDNA of ∆6-desaturase from R. oryzae Mycelia were harvested by filtration and washed with phosphate-buffered saline buffer. The mycelia were frozen in liquid nitrogen and ground with mortar and pestle into a fine powder. Total RNA was extracted from the powder according to the method of Chomczynski [6] based on guanidinium thiocyanate, and stored at –80 °C for future use. The GeneRacer kit from Invitrogen (USA) was used for the cloning of the full-length Δ6-desaturase cDNA from R. oryzae through rapid amplification of cDNA ends (RACE). NCBI nucleotide sequences of fungal Δ6-desaturases from Rhizopus oryzae, Rhizopus nigricans (GenBank acc. nos. AY583316 and AY795076) and Rhizopus arrhizus (AY320288) were multi-aligned using the Vector NTI Suite 9.0. A pair of PCR primers, FD6O (5′-AAGGTGTACGATGTGACTGAATTCGT-3′) and RD6O (5′-TGCTCGATTTGATAGTTCAATCCACC-3′), which cor-respond to two conserved sites of the fungal Δ6-de-saturase genes, were designed and synthesized (Shang-hai Sangon Biotechnology Corporation Ltd., Shanghai, China) to amplify the conserved region of the target gene. Of Taq polymerase (Promega, USA), 2.5 U was used in the 50 μl reaction mixture, using 1 μl first-

strand cDNA as template. PCR was performed on a Biometra Tgradient thermal cycler (Biometra GmbH, Göttingen, Germany) with predenaturation for 3 min at 96 °C, followed by 36 cycles of amplification (1 min at 96 °C, 1 min at 57 °C, and 1 min at 72 °C) and a final extension at 72 °C for 8 min. After 1.0% agarose gel electrophoresis with ethidium bromide detection, the target DNA band was recovered and ligated with the pMD18-T vector (TaKaRa, Dalian, China) for transforma-tion into the E. coli strain DH5α. Based on the sequences of conserved regions, four gene-specific PCR primers were designed for the amplification of the cDNA ends: two forward primers, FD6O3-1 (5′-GCTTTGAACCACAAT GGTATGCCTG-3′) and FD6O3-2 (5′-GGGTGATTGGTTCAT GGGTGGATTG-3′), and two reverse primers, RD6O5-1 (5′-GTTCCAAGTGCGGTCTTCGAAGCAT-3′) and RD6O5-2 (5′-ATTGATCCCGAAGCTGACGCATTTC-3′). FD6O3-1 was paired with the kit primer 3P while FD6O3-2 was paired with the kit primer 3NP, for the primary and nested PCR reactions of 3′ RACE, respectively. In 5′ RACE, RD6O5-1 and RD6O5-2 were paired with the kit primers 5P and 5NP, for the primary and nested PCR reactions, respectively. All four PCR reactions were run under the same cycling conditions: predenaturation at 96 °C for 3 min, followed by 30 cycles of amplification (1 min at 96 °C, 1 min at 60 °C, and 40 s at 72 °C), and then 8 min at 72 °C. The forward primer FRoD6O (5′-ATGAGTA CATCAGATCGTCAATCAG-3′) and the reverse primer RRoD6O (5′-TTAAAATGACTTTTTGCTCAATTGCAA-3′), corresponding to the 5′ and 3′ cDNA ends, respectively, were designed to amplify the RoD6D full-length cDNA and the corresponding genomic sequence under the following cycling conditions: predenaturation at 96 °C for 3 min, followed by 36 cycles of amplification (1 min at 96 °C, 1 min at 60 °C, and 2 min at 72 °C), and then 8 min at 72 °C. DNA recovery, subcloning and sequenc-ing of the cDNA ends and the full-length gene frag-ments were performed by the methods described above. Sequence alignments, ORF translation and molecular mass calculation of the predicted protein were carried out on Vector NTI Suite 9.0. GenBank BLAST searches were done on the NCBI website (http:// www.ncbi.nlm.nih.gov/), while structural analysis of the predicted RnD6D protein was carried out on online software linked by websites (http://www.expasy.org).

Genetic engineering experiments Two specific primers, FRoD6O (5′-CTGCCTCGAGATGA GTACATCAGATCGTCAATCAG-3′) and RRoD6O (5′-CTGC TTCGAATTAAAATGACTTTTTGCTCAATTGCAA-3′), were designed to amplify the full-length coding region of the RoD6O cDNA. In order to facilitate subsequent subclon-

Journal of Basic Microbiology 2012, 52, 1–5 γ-Linolenic acid ∆6-fatty acid desaturase gene R. oryzae 3


ing, XhoI and HindIII restriction sites (underlined) were incorporated into the 5′ ends of FRoD6O and RRoD6O, respectively. The amplified cDNA was first cloned into pMD18-T and then transformed into DH5α for sequenc-ing. The ORF fragment identified by sequencing was cut out from the recombinant pMD18-T using XhoI and HindIII double digestion and recovered to be ligated with the XhoI- and HindIII-digested yeast expression vector pYES2.0 (Invitrogen, USA), to generate the re-combinant plasmid pYRnD6D. S. cerevisiae was trans-formed with pYRnD6D and pYES2.0, respectively, using the lithium acetate method. Transformants were se-lected by plating on synthetic minimal medium agar lacking uracil (SC-Ura) and grown at 30 °C for 3 d. Ex-pression of RoD6O was under the transcriptional control of the inducible GAL1 promoter. Yeast cultures were grown to the logarithmic phase at 30 °C in synthetic minimal medium (SC-Ura) containing 2% raffinose and 0.67% yeast nitrogen, supplemented with 0.4 mM LA (Sigma). The cells were induced by the addition of 2.5% galactose and cultivated for a further 48 h at 25 °C. The cells were harvested by centrifugation, washed three times with double-distilled water, dried and ground with mortar and pestle into a fine powder.

Fatty acid analysis Yeast powder (150 mg) was disrupted by mortar and pestle, mixed with 5 ml ether/chloroform (1:1) and incubated at room temperature for 16 h for the thor-ough extraction of the cellular fatty acids. Then the extracts were subjected to saponification by adding 5 ml 5% KOH methanol solution. The fatty acid methyl esters (FAME) were subsequently analyzed by gas chro-matography (GC; GC-2010, Shimadzu, Kyoto, Japan). A fused capillary column NUKOL 30 m × 0.25 mm (Su-pelco) was used in this research, at a column tempera-ture of 170 °C, an injection temperature of 250 °C, and a detector temperature of 250 °C. The mass spectrum of a new peak was compared with that of the standards (Sigma) for fatty acid identification.

Results

Cloning of the R. oryzae ∆6-fatty acid desaturase gene The amplification of a conserved region of RoD6D using first-strand cDNA as template resulted in a DNA band that was proved to be of 881 bp in length by sequenc-ing. An NCBI BlastX search of this fragment showed broad homologies to Δ6-desaturases from fungi. Ampli-fication using the primer pair FRoD6D/RRoD6D and

total cDNA as template resulted in a DNA band, and the sequenced full-length cDNA of RoD6D (without polyA tail) was 1176 bp long, which is completely in consensus with the putative full-length cDNA. The FRoD6D/RRoD6D primer pair was also used to amplify the genomic DNA template of R. oryzae to characterize the intron/exon structure of this gene. It yielded a bright DNA band, which was proved to be 1565 bp long by sequencing. Alignment of the full-length cDNA and the corresponding genomic sequence resulted in five gaps within the cDNA sequence lane, indicating that this gene has five introns. When the five introns were deleted from the genomic sequence, the resulting se-quence completely coincided with the full-length cDNA of RoD6D, suggesting that we have successfully isolated both the mRNA and the genomic sequences of this gene. The mRNA and corresponding genomic nucleo-tide sequences of RoD6D were deposited in the NCBI GenBank under the accession numbers JQ063116 and JQ063117, respectively. A nucleotide-nucleotide BLAST (blastn) search of the RoD6D full-length cDNA on the NCBI website showed 74–74.6% identity in the local alignment to the R. oryzae Δ6-fatty acid desaturase gene (AY583316) and the R. stolonifer Δ6-fatty acid desaturase mRNA (AY795075). The ORF of the RoD6D mRNA encoded a polypeptide of 391 amino acid residues, with a calculated Mw of 44.28 kDa and a pI of 6.79. Comparison of the deduced amino acids of D6DR with other fungal Δ6-fatty acid desaturases revealed four conserved histidine-rich mo-tifs at amino acid positions 49, 156, 191, and 328 (Fig. 1). The deduced amino acid sequence of D6DR was compared with those of desaturases from other organ-isms. The results showed that this sequence had 66% identity to the Δ6-fatty acid desaturase from R. oryzae and 63% identity to that from R. stolonifer. A protein-protein BLAST (blastp) search of the RoD6D deduced amino acid sequence showed very wide homology to desaturases from fungi (Fig. 2).

Figure 1. Deduced amino acid sequence of the ∆6-desaturase from the fungus R. oryzae DR3. A cytochrom b5-like domain and three conserved histidine-rich motifs are underlined.

4 Yu Zhu and Bi-Bo Zhang Journal of Basic Microbiology 2012, 52, 1–5


Figure 2. Phylogenetic relationship between RoD6D and Δ6-desaturases from various fungi. AF465282 Mortierella isabellina, AB052086 Mucor circinelloides, AY320288 Rhizopus sp. NK030037, JQ063116 Rhizopus oryzae, AY795076 Rhizopus stolonifer, DQ291156 Rhizopus stolonifer strain YF6, AY583316 Rhizopus oryzae.

All the above clues strongly suggested that the RoD6D gene reported here encoded a putative Δ6-desaturase involved in the synthesis of GLA in R. oryzae.

Functional analysis of the R. oryzae ∆6-fatty acid desaturase Fatty acid analysis indicated that exogenous LA was incorporated into the lipids of the transformed yeast. GC analysis of the FAME revealed that a novel fatty acid peak corresponding to the GLA methyl ester standard was detected in the yeast transformed with pYRnD6D, which was absent in the yeast containing the empty vector pYES2.0. The percentage of this new fatty acid was 14.24% of the total fatty acids. These results showed that pYRnD6D encoded a Δ6-fatty acid desatu-rase. The expressed enzyme specifically converted the incorporated LA (0.4 mM) to GLA (Fig. 3). Regulation of desaturase gene expression has been investigated in various organisms, and temperature was shown to be acrucial factor that influences the gene expression [7]. These results suggested that RnD6D was likely to play a

role for R. oryzae in acclimatizing to low temperature, which could be attributed to an improvement in mem-brane fluidity by increasing the degree of desaturation of the cellular fatty acids.

Discussion

GLA is a PUFA in the human body with many medicinal and health-promoting effects; it can be used in cosmet-ics and medicine [8, 9]. The main sources of GLA are plant seeds and fungi from fermentation, with the type of fermentation (solid- versus submerged-state fermen-tation) affecting the production of metabolites [10–12]. However, it is obvious that GLA production from the current sources is inadequate for supplying the expand-ing market, due to such significant problems as low productivity, insecurity, expensive downstream proc-essing, etc. [13]. Δ6-Fatty acid desaturase genes have been previously cloned and characterized from several fungi, such as Rhizopus nigricans and R. arrhizus [14, 15], but fungal sources would bring about new issues of GLA safety. Nowadays, consumers are demanding for foods with an increasing range of properties, such as pleasant flavor, low calorie value or low fat content, and beneficial health effects. In our previous work, we isolated the Rhizopus oryzae strain DR3 from the fermen-tation of Douchi. Douchi is a popular oriental fermen-tation food in China. Lu et al. [16] report on the produc-tion of GLA from Douchi fermentation. Using GC analysis, we discovered that this species also synthe-sizes PUFA, especially GLA. So, gene isolation and func-tional characterization of Δ6-fatty acid desaturases are a prerequisite for the elucidation of the related molecu-

Figure 3. Identification of GLA in transgenic S. cerevisiae by GC analysis, with GLA as the internal standard. (A) S. cerevisiae transformed with the control vector pYES2.0. (B) S. cerevisiae transformed with the recombinant plasmid pYRoD6D. The arrowhead indicates the novel peak of GLA.

Journal of Basic Microbiology 2012, 52, 1–5 γ-Linolenic acid ∆6-fatty acid desaturase gene R. oryzae 5


lar mechanism and the development of applicable prod-ucts. We first cloned and functionally characterized a Δ6-fatty acid desaturase gene involved in GLA biosynthesis in R. oryzae from Douchi, one kind among many tradi-tional Chinese foods consisting of fermented soybeans. So the strain of R. oryzae DR3 is safe. Knowledge of the enzymatic reactions and the corresponding metabolic pathways, as well as the growing number of desaturase genes cloned from different organisms, allows the for-mulation of some starting hypotheses and provides a basis for the interpretation of the biosynthesis net-works of PUFA. By overexpression of RoD6D in bioreac-tors, such as fungal cultures or oil-producing crops, we can expect a high-output production of GLA.

Acknowledgements

This research was supported by the Key Project of the Chinese Ministry of Education (200202128), China Na-tional “948” Program（2003Q04）and China National “863” Program, etc.

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((Funded by • Key Project of the Chinese Ministry of Education; grant number: 200202128 • China National “948” Program; grant number 2003Q04 • China National “863” Program))

molecular cloning and functional characterization of a Δ6-fatty acid desaturase gene from rhizopus...

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