Full Length Research Paper
Screening and molecular identifying overproducing Gamma linolenic acid fungi and cloning Delta 6-desaturase gene
He Lu1
Yu Zhu2
1: Department of Microbiology, Chongqing Medical University, Chongqing 400016, People's Republic of
China 2: Department of Immunology, Chongqing Medical University, Chongqing 400016, People's Republic of China
Running title: Molecular identifying, Cloning, Delta 6-desaturase, GLA
Key words: Screening,
1
GLA, △6-fatty acid desaturase gene,
Cloning
Corresponding author: he Lu, Chongqing 400016, People's Republic of China.
E-mail:[email protected]; Tel: +86-23-68023165; This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bab.1281. This article is protected by copyright. All rights reserved.
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Abstract This research aims at isolating and identifying the Gamma linolenic acid producing fungi in the traditional Chinese salt-fermented soybean food, Douchi, from Yongchuan, China. In this study Rhizopus oryzae DR3 was identified as a novel fungal species that produces large amounts of γ-linolenic acid. A full-length cDNA, designated her as RoD6D, with high homology to fungal △6-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 was 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 △6-fatty acid desaturase activity that led to the accumulation of γ-linolenic acid. The results show that Douchi contains a large natural of diverse composition, and some strains could be selected as starters for functional fermented foods. This study has also laid a foundation on developing functional Douchi products for further research.
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Introduction Gamma linolenic acid (GLA 18:3, △6,9,12)is an important n-6 polyunsaturated fatty acids (PUFAs) that has played important role as structural components of membrane lipids (Needleman et al., 1986; Vrinten et al., 2007), and further metabolized to produce eicosanoids such as leukotrienes. So, it has many healthy effects and medicinal, such as have selective tumoricidal action and in anti-Inflammatory (Shinji et al., 2010; Kim, 2012). But, GLA is rarely found in common food and is present in trace amounts in some seed oil plants and nut (Hansson et al., 1989; De et al., 2004). Therefore, this is an important way of microbial fermentation get GLA. However, the production of fermentation is inadequate for supplying the expanding market due to the low productivity. Therefore, through the screening and selection for obtain high yield strains GLA accumulating GLA, or recompose produce desired oil in transgenic microorganisms. Because, △6-fatty acid desaturase catalyzes the conversion of linoleic acid (LA, 18:2, △9,12) and α-linolenic acid (18:3, △9,12,15) to GLA (18:3, △6,9,12) and stearidonic acid (18:4, △6,9,12,15) respectively. So, Many △6-fatty acid desaturase gene has been previously cloned and characterized from several fungi, such as Mortierella alpine, Rhizopus. nigricans (Chen et al., 2005). But，looking for more excellent, safe GLA-producing bacteria, is still one of the hot current research. Douchi, one kind among many traditional Chinese foods, is consisted of fermented soybeans. The soybean paste is rich in food microorganisms, such as Bacillus. subtilis, Bacillus amyloliquefaciens, and Mucorales (Chen et al., 2011). The microorganisms taking place during fermentation can create peptides and free amino acids and some fungi possess an impressive metabolic diversity, so it may have different flavors and nutrient and chemical compositions (Tomasini
et al., 1997; Stahnke et al., 2002; Feron et al., 1996). The show that
Douchi contains a large natural of diverse composition, and some strains could be selected as starters for functional fermented foods. Rhizopus oryzae is used in the food industry and its products are generally recognised as safe food. So, if R. oryzae is isolated from fermentation Douchi, identification of the genes associated with the synthesis of GLA will contribute to characterizing the desaturase genes in R. oryzae, establishing a simple model for studying metabolic pathways of GLA in eukaryotes and provide the primary basis for the future application of gene engineering to GLA production. If Douchi contains GLA it may be directly absorbed through diet. Thus, a low cost of GLA production process could be achieved in fermented Douchi. In this work, we report the cloning and expression of a △6-desaturase of R. oryzae strain DR3 from Douchi. This study has also laid a foundation for further research on developing functional Douchi products. This article is protected by copyright. All rights reserved.
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Materials and methods Collection of samples and Chemicals Ten Douchi samples were collected from the main Douchi-producing areas of Yongchuan, China. Each 150g sample, prepared by using traditional household methods, was collected and put into a sterilized sampling bag, then transported to the laboratory where it was analysed. GLA were purchased from Sigma-Aldrich (St Louis, MO, USA). Bacto-yeast extract, 2% bacto-peptone and other important medium components were purchased from Oxoid Ltd (Hampshire, England). Saccharomyces cerevisiae strain INVScl was used as recipient in transformation experiments and was grown at 30℃ in complex medium (Yeast Extract Peptone Dextrose, YPD) containing 1% bacto-yeast extract, 2% bacto-peptone and 2% glucose. 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 by using a Thermal cycler from Eppendorf. Isolation and culture of the Gamma linolenic acid producing fungi Douchi were sampled from different Pottery fermentation tanks. First, samples were serially diluted 5-fold with distilled water, 10μL drops of each dilution were separately plated in potato dextrose agar (PDA) to which 0.1% (W/V) gentamicin was added and then screened by using Sudan black staining. These plates were incubated at 26 ± 1℃ for 5 days. Pure fungal cultures were obtained. The strains were preserved on PDA slants and stored at 4℃. Morphological identification of the fungi The strain was cultured on PDA plates at 26℃ for 5 days. If we observed the colonial morphology, e.g., shape, size, color and odor, we could describe spore in great details, and the microscopic features were measured from strains grown on PDA plates 2 days. Polyunsaturated fatty acids analysis The fatty acid methyl esters (FAME) were prepared according to the method of Morrison and Smith (Morrison and Smith, 1964). Mycelia powder (150 mg) was disrupted by mortar and pestle, mixed with 5 mL aether:chloroform (1:1) and incubated at room temperature for 16h for the thorough extraction of cellular fatty acids. Then the extracts were subjected to saponification by adding 5 mL 5% KOH methanol solution. The FAME were subsequently analyzed by gas chromatography (GC; GC-2010, Shimadzu, Kyoto, Japan). A fused capillary column NUKOL 30 m × 0.25 mm Supelco was used in this research, at a column temperature of 170℃,
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an injection temperature of 250℃, and a detector temperature of 250℃. The mass spectrum of a new peak was compared with that of the standards (Sigma) for fatty acid identification. Molecular identifying of the Strain The mycelia were grown in 500 ml Erlenmeyer flasks containing 200 ml of potato dextrose broth with 100 rpm for 5 days at 26±1℃. The mycelia were harvested by filtration and grinded with liquid nitrogen thoroughly. Total genomic DNA was extracted by using the method suggested by the instructions in the Genomic Prep Fungal
DNA
midi
kit
(Omega
Bio-Tek,
Inc,
Norcross,
GA,
USA).
Primers
ITS1
(5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) was used for amplification and sequence of 18S rRNA gene. We used a polymerase chain reaction (PCR) protocol with a thermostable DNA polymerase (Takara, Japan) and cut out the amplified band about 600 bases in 1% TBE-agarose gel and then extracted it using Gene Clean Turbo (Qbiogene, Baton Rouge, LA, USA), and Sequenced in the ABI PRISM 77 DNA sequencer. The strain sequences of 18S rDNA were used as query sequence to search for similar sequences from GenBank and used for subsequent phylogenetic analyses. Cloning and Genetic engineering experiments of △6-desaturase gene 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 (Chomczynski and Sacchi 1987) based on guanidinium thiocyanate and stored at -80℃ for future use. The GeneRacer kit from Invitrogen (USA) was used for the cloning of full-length △6-desaturase cDNA of the
through
rapid
amplification
of
cDNA
ends
(5’-AAGGTGTACGATGTGACTGAATTCGT-3’)
(RACE).
A
pair
of
PCR
primers
and
FD6O RD6O
(5’-TGCTCGATTTGATAGTTCAATCCACC-3’), which corresponded to two conserved sites of fungal △ 6-desaturase genes, were designed and synthesized (Shanghai Sangon Biotechnology Corporation Ltd, Shanghai, China) to amplify the conserved region of the target gene. PCR amplification was performed according to the method of Lu et al (Lu et al., 2009). Sequence alignments, ORF translation and molecular mass calculation of predicted protein were carried out on Vector NTI Suite 9.0. GenBank BLASTs were done on NCBI (http://www.ncbi.nlm.nih.gov/), while structural analysis of predicted RoD6D protein was carried out on online softwares linked by websites (http:// www.expasy.org). The ORF fragment identified by sequencing was cut out from the recombinant pMD18-T using XhoI and
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HindIII double digestion and recovered to be ligated with the XhoI and HindIII digested yeast expression vector pYES2.0 (Invitrogen, USA) to generate the recombinant plasmid pYRoD6D. S. cerevisiae was transformed with pYRoD6D and pYES2.0 respectively by using the lithium acetate method (Lu et al., 2009). All experiments were conducted three times with three replicates of each isolate. The data are interpreted as mean±SD (n=9).
Results Isolation and Identification of the Gamma linolenic acid producing fungi In order to obtain high yield strain of GLA, the strain accumulating GLA was isolated by using Sudan black staining. Firstly, PDA plate was used for isolation of fungal, thirty strains in PDA plates were selected and then Sudan black was used to stain the strains. The darker strains were selected, indicating that the Strains for potential of GLA produced strains. The mycelia Fatty acids were transformed to methyl esters and analyzed by gas chromatography (GC) (Fig 1). GLA standards were used as reference for the retention times. According to these characteristics described above, strain DR3 was screening. The strain DR3 showed a content of GLA in total lipid of 16.68%, compared with another known as strains production of GLA we screened the strains DR3 with higher production capacity. (Table 1) The strains colonies reached 3–5 cm diameter on PDA after 5 days, color variable with state of sporulation grew from white to grayish and rhizoids were well developed. Sporangiospore arose from basal hyphae. According to these characteristics described above, strain DR3 was identified as one certain species of Rhizopus. Meanwhile, the 18S rDNA sequence of strain DR3 was also analyzed. In the NCBI GenBank under the accession numbers KC571633. The sequence with 623bp showed 100% identity with that of Rhizopus oryzae strain CNM-CM-6256(JX120700). In the light of the 18S rRNA genes sequences of strain DR3 and other 6 representative reference taxa, the Neighbor-joining tree revealed distinctly that fungus DR3 was as a member of the Rhizopus clade (Fig. 2). According to the phylogenetic tree based on related fungal, the strain DR3 was highly similar to Rhizopus oryzae, which was identical with the result of morphological identification Therefore, the strain DR3 was identified as Rhizopus oryzae. Cloning of the R. oryzae DR3 △6-fatty acid desaturase gene A cDNA fragment was proved to be of 881 bp in length by sequencing. NCBI Blastx search of this fragment showed broad homologies to △6-desaturases from fungi. The sequenced full-length cDNA of RoD6D (without poly A tail) was 1176bp long, which is completely in consensus with the putative full-length cDNA. The primer pair was also used to amplify the genomic DNA template of R. oryzae strain DR3 to characterize the This article is protected by copyright. All rights reserved.
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intron/exon structure information of this gene. It yielded a bright DNA band which was proved to be 1565bp 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 sequence completely coincided with the full-length cDNA of RoD6D, which suggested that we have successfully isolated both the mRNA and the genomic sequences of this gene. The mRNA and corresponding genomic nucleotide sequences of RoD6D were deposited in the NCBI GenBank under the accession numbers JQ063116 and JQ063117 respectively. Sequence comparison and structure prediction 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. A BLAST analysis using the D6DR amino acid sequence showed the similarity with other PUFA of the desaturase in the GenBank database. A multiple sequence alignment of this sequence against similar desaturase (Fig.3) the cytochrome b5 domain conserved sequence ‘‘HPGG’’ and the three histidine-box sequences ‘‘HDFGH’’, ‘‘HNTHH’’ and ‘‘QIEH’’ of the desaturase domain. On NCBI local alignment, RoD6D showed 63% identities and 64% positives to △ 6-desaturases from R. oryzae (AAS93682) and R.stolonifer(AAX22052), but less than 31% identities and 61% positives to all other desaturases. Phylogenetic analysis also demonstrated the homology between RoD6D and △6-desaturases from various organisms (Fig 4). Signal P V2.0 prediction did not detect any signal peptide sequence in RoD6D. TMHMM prediction yielded 4 potential transmembrane helices in RoD6D (Fig. 5). Confirmation of the activity of the △6-fatty acid desaturase 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 pYRoD6D, 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 pYRoD6D encoded a △-6 fatty acid desaturase. The expressed enzyme specifically converted the incorporated LA (0.4mM) to GLA (Fig.6). Comparison of different production methods Currently on the market GLA is commodity obtained from seeds of plant and microorganisms source. But, in Table 2, we can clearly see that fermentation Douchi production of GLA is easy, compared with other methods.
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Discussion Dietary polyunsaturated fatty acids are indispensable for human cells to maintain their structure and function (Calder and Newsholme, 1992). GLA is a PUFA with many medicinal and healthy promoting effects, such as kill cancer cell and anti-infection (Gill and Valivety, 1997; Raclot et al., 1997). The main sources of GLA are plant seeds such as black currant and fungi from fermentation, like species of Mortierella, and Mucor are known (Kapoor and Huang, 2006; Kenny et al., 2000; Sakuradani et al., 2009). However, it is obvious that GLA production from the current sources is inadequate for supplying the expanding market due to such significant problems as low productivity, insecurity, expensive downstream processing and etc (Wan et al., 2009). So, this is still the hot spot of to find more excellent GLA producing strain. Many species of the GLA producing fungal have been isolated, such as Mortierella alpine, Rhizopus stolonifer, Mucor. However, it has fewer reports that R. oryzae has not been used as one kind of producin GLA fungal in the Douchi. In order to obtain high yield strain GLA with Sultan black staining is a good screening method. Strain DR3, which is able to produce abundant GLA, was isolated from the Douchi by using Sudan black staining. For further research of this strain DR3 was identified. Morphological identification which employed light microscope showed that DR3 was Rhizopus sp.18S rDNA gene were then cloned sequences proved that strain DR3 was R. oryzae. In this study, the R. oryzae in spontaneously fermented Douchi was studied by conventional culture-dependent methods combined with molecular biological methods.
Although the GLA can be got in different biosynthesis pathways of strains (Laho et al., 2011; Certik et al., 2006; Zhang et al., 2004), and △6-fatty acid desaturase genes has been previously cloned and characterized from several fungi, such as Rhizopus nigricans, R.arrhizus (McCue and Shetty,2003; Fujita et al.,2001; Fujita et al.,2003). We have cloned and confirmed of the activity a new △6-fatty acid desaturase gene involved in GLA biosynthesis in strain DR3. This enzyme contains the signature desaturase motif four membrane domains, and share extensive similarity in secondary structure arrangement. It suggests that they retained the correct conformation and intracellular location. However, they share less identity to other △6-fatty acid desaturase such as the △6-fatty acid desaturase from R. oryzae AAS93682 (63 % identity). The percentage of GLA was 16.68% of the total fatty acids. This result showed that some special characterization of △6-fatty acid desaturase of R. oryzae DR3. It also shows that △6-fatty acid desaturase activity may be enhanced by various effects, For example, the codon optimization or the length of sequence to further improve the level of expression obtained (Xue et al., 2013; Lu et al., 2013; Song et al., 2013). However, fungal sources would bring about new issues of GLA safety. So, how to reduce the risk of food pollution is important, and direct consumption of This article is protected by copyright. All rights reserved.
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natural foods containing GLA will best meet the needs of people. Nowadays, much attention has been paid by consumers towards natural bioactive compounds as functional ingredients in the diet. Although the GLA can be got in different biosynthesis pathways and strains (Laho et al., 2011; Certik et al., 2006; Zhang et al., 2004), but direct consumption of natural foods containing GLA will best meet the needs of people. With the growing interest in traditional soybean foods, the functional ingredients (e.g., isoflavone, aqueous extract, and aroma components) contained in Douchi has been fully investigated (Hesseltine and Wang 1972; Chaiyasut et al., 2010). The recent study indicates that Douchi is useful to relieve tiredness, weakness, insomnia, and poor appetite (Zhang et al 2004), and is able to prevent and cure diabetic (Chen et al., 2007; McCue et al., 2003; Fujita et al., 2001). However, preliminary experiment of GLA in commercial Douchi from local supermarkets sample did not contain GLA. So, we isolated strain DR3 of R. oryzae from Douchi. According to the phylogenetic tree based on related fungal, the strain DR3 was identified as R. oryzae. By using GC analysis we have discovered that this species also synthesizes PUFA, especially GLA. R. oryzae is used in the food industry and its products are generally recognised as safety. Therefore gene isolation and functional characterization of △6-fatty acid desaturases from R. oryzae are a prerequisite for the elucidation of the related molecular mechanism and the development of applicable products. The bioactivity of the PUFA produced by strain DR3, R. oryzae, will be further studied and the results will be published in the future. This study has also laid a foundation for further research on developing functional Douchi products. It also shows that Douchi contains a large natural diverse composition and some strains could be selected as starters for functional fermented foods. Confirmed This research was supported by the Key Project of Chinese Ministry of Education（200202128）, China National “948” Program（2003Q04） Acknowledgements This research was supported by the Key Project of Chinese Ministry of Education（200202128）, China National “948” Program（2003Q04）.
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and Sidney J. Circle(eds.), Westport, Conn.USA. Gill, I., and Valivety, R. (1997). Polyunsaturated fatty acids, Part 1: Occurrence, biological activities and applications. Trends biotechnol 15, 401-409 Kapoor, R., Huang, Y.S. (2006). Gamma linolenic acid: an antiinflammatory omega-6 fatty acid. Curr Opin Lipidol 7,531-535. Kenny, F., Pinder, S., Ellis, I., Gee, J., Nicholson, R., Bryce, R., et al. (2000). Gamma linolenic acid with tamoxifen as primary therapy in breast cancer. Int J Cancer 85,643–648
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Song, L.Y., Lu, W.X., Hu, J., Yin, W.B., Chen, Y.H, Wang, B.L, et al. (2013). The role of C-terminal amino acid residues of a Δ⁶ -fatty acid desaturase from blackcurrant. Biochem Biophys Res Commun 431,675-679 Stahnke, L.H, Holck, A.L, Jensen, A., Nilsen, A., Zanardi, E. (2002). Maturity acceleration by Staphylococcus carnosus in fermented sausage–relationship between maturity and flavor compounds. J Food Sc 67,1914–1921 Tomasini, A., Fajardo, C., Barrios-Gonza´lez, J. (1997). Gibberellic acid production using different solid-state fermentation systems. World Journal of Microbiology and Biotechnology 13,203–206 Vrinten, P., Wu, G., Truksa, M., and Qiu, X. (2007). Production of polyunsaturated fatty acids in transgenic plants. Biotechnol Genet Eng Rev 24, 263-279 Wan, X., Zhang, Y.B., Wang, P., and Jiang, M.L. (2009). Production of gamma-linolenic acid in Pichia pastoris by expression of a delta-6 desaturase gene from Cunninghamella echinulata. J Microbiol Biotechnol 19,1098-1102 Xue, Z., He, H., Hollerbach, D., Macool, D.J, Yadav, N.S, Zhang, H., et al. (2013). Identification and characterization of new Δ-17 fatty acid desaturases. Appl Microbiol Biotechnol 97,1973-1985 Zhang, Q., Li, M., Ma, H., Sun, Y. (2004). Identification and characterization of a novel delta 6-fatty acid desaturase gene from Rhizopus arrhizus. FEBS Lett 556:, 81-85
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Fig. 1 Fatty acid composition of R. oryzae DR3 by GC analysis (Red line is standards of GLA)
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Fig. 2 Neighbor-joining tree of Fungal R. oryzae DR3 (KC571633) based on 18S rRNA gene sequences. Confidence values above 100% obtained from a 1000-replicate bootstrap analysis are shown at the branch nodes. Bootstrap values from neighbor-joining method were determined. Mucor fragilis (JN198474) and Mucor plumbeus strain UASWS0823 (JX139728) were used as the out group.
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Fig. 3 Sequence alignment of deduced amino acids of delta 6-desaturase from fungus R. oryzae DR3 (ROD6) with those of R. delemar (RDD6), R. stolonifer (RSD6), Mortierella alpine (M. alpine ). A cytochrom b5-like domain and three conserved histidine-rich motifs are underlined.
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Fig. 4 A Phylogenetic relationship between RoD6D and △6-desaturases from various fungi. ADD50000 Sparus aurata, AAC49700 Borago officinalis. ADI49410 Phaeodactylum tricornutum, ADE06661 Mortierella alpina, AAX22052 Rhizopus stolonifer, ADX21039 Mucor circinelloides f. lusitanicus, AAS93682 Rhizopus oryzae. ADX21038 Mucor fuscus. AFD18733 Rhizopus oryzae DR3
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Fig. 5 Hydropathy profile of RoD6D of R.oryzae DR3
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Fig. 6 Identification of GLA in transgenic S. cerevisiae by GC analysis A: S. cerevisiae transformed with control vector pYES2.0. B: S. cerevisiae transformed with the recombinant plasmid pYRoD6D. The arrowhead indicates the novel peak of GLA
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Table:1 Relative percentage of fatty acid and GLA of fungal Strain
Total fatty acids
GLA (% of total fatty acids)
Cunninghamella echinulata
36.73
6.4
Mucor rouxii
31.25
15.42
Rhizopus nigricans
54.83
24.32
DR3
41.25
16.68
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Table:2
Compared with other methods
Sample
New technology Douchi
Supermarket Douchi
Fungal lipid
GLA in total fatty acids
3.54±0.36
0.00
16.68
Food
yes
yes
No
Purification
No need
No need
Need
(%)
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Screening and molecular identifying overproducing Gamma linolenic acid fungi and cloning Delta 6-desaturase gene
He Lu1
Yu Zhu2
1: Department of Microbiology, Chongqing Medical University, Chongqing 400016, People's Republic of
China 2: Department of Immunology, Chongqing Medical University, Chongqing 400016, People's Republic of China
Running title: Molecular identifying, Cloning, Delta 6-desaturase, GLA
Key words: Screening,
1
GLA, △6-fatty acid desaturase gene,
Cloning
Corresponding author: he Lu, Chongqing 400016, People's Republic of China.
E-mail:[email protected]; Tel: +86-23-68023165; This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bab.1281. This article is protected by copyright. All rights reserved.
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Abstract This research aims at isolating and identifying the Gamma linolenic acid producing fungi in the traditional Chinese salt-fermented soybean food, Douchi, from Yongchuan, China. In this study Rhizopus oryzae DR3 was identified as a novel fungal species that produces large amounts of γ-linolenic acid. A full-length cDNA, designated her as RoD6D, with high homology to fungal △6-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 was 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 △6-fatty acid desaturase activity that led to the accumulation of γ-linolenic acid. The results show that Douchi contains a large natural of diverse composition, and some strains could be selected as starters for functional fermented foods. This study has also laid a foundation on developing functional Douchi products for further research.
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Introduction Gamma linolenic acid (GLA 18:3, △6,9,12)is an important n-6 polyunsaturated fatty acids (PUFAs) that has played important role as structural components of membrane lipids (Needleman et al., 1986; Vrinten et al., 2007), and further metabolized to produce eicosanoids such as leukotrienes. So, it has many healthy effects and medicinal, such as have selective tumoricidal action and in anti-Inflammatory (Shinji et al., 2010; Kim, 2012). But, GLA is rarely found in common food and is present in trace amounts in some seed oil plants and nut (Hansson et al., 1989; De et al., 2004). Therefore, this is an important way of microbial fermentation get GLA. However, the production of fermentation is inadequate for supplying the expanding market due to the low productivity. Therefore, through the screening and selection for obtain high yield strains GLA accumulating GLA, or recompose produce desired oil in transgenic microorganisms. Because, △6-fatty acid desaturase catalyzes the conversion of linoleic acid (LA, 18:2, △9,12) and α-linolenic acid (18:3, △9,12,15) to GLA (18:3, △6,9,12) and stearidonic acid (18:4, △6,9,12,15) respectively. So, Many △6-fatty acid desaturase gene has been previously cloned and characterized from several fungi, such as Mortierella alpine, Rhizopus. nigricans (Chen et al., 2005). But，looking for more excellent, safe GLA-producing bacteria, is still one of the hot current research. Douchi, one kind among many traditional Chinese foods, is consisted of fermented soybeans. The soybean paste is rich in food microorganisms, such as Bacillus. subtilis, Bacillus amyloliquefaciens, and Mucorales (Chen et al., 2011). The microorganisms taking place during fermentation can create peptides and free amino acids and some fungi possess an impressive metabolic diversity, so it may have different flavors and nutrient and chemical compositions (Tomasini
et al., 1997; Stahnke et al., 2002; Feron et al., 1996). The show that
Douchi contains a large natural of diverse composition, and some strains could be selected as starters for functional fermented foods. Rhizopus oryzae is used in the food industry and its products are generally recognised as safe food. So, if R. oryzae is isolated from fermentation Douchi, identification of the genes associated with the synthesis of GLA will contribute to characterizing the desaturase genes in R. oryzae, establishing a simple model for studying metabolic pathways of GLA in eukaryotes and provide the primary basis for the future application of gene engineering to GLA production. If Douchi contains GLA it may be directly absorbed through diet. Thus, a low cost of GLA production process could be achieved in fermented Douchi. In this work, we report the cloning and expression of a △6-desaturase of R. oryzae strain DR3 from Douchi. This study has also laid a foundation for further research on developing functional Douchi products. This article is protected by copyright. All rights reserved.
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Materials and methods Collection of samples and Chemicals Ten Douchi samples were collected from the main Douchi-producing areas of Yongchuan, China. Each 150g sample, prepared by using traditional household methods, was collected and put into a sterilized sampling bag, then transported to the laboratory where it was analysed. GLA were purchased from Sigma-Aldrich (St Louis, MO, USA). Bacto-yeast extract, 2% bacto-peptone and other important medium components were purchased from Oxoid Ltd (Hampshire, England). Saccharomyces cerevisiae strain INVScl was used as recipient in transformation experiments and was grown at 30℃ in complex medium (Yeast Extract Peptone Dextrose, YPD) containing 1% bacto-yeast extract, 2% bacto-peptone and 2% glucose. 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 by using a Thermal cycler from Eppendorf. Isolation and culture of the Gamma linolenic acid producing fungi Douchi were sampled from different Pottery fermentation tanks. First, samples were serially diluted 5-fold with distilled water, 10μL drops of each dilution were separately plated in potato dextrose agar (PDA) to which 0.1% (W/V) gentamicin was added and then screened by using Sudan black staining. These plates were incubated at 26 ± 1℃ for 5 days. Pure fungal cultures were obtained. The strains were preserved on PDA slants and stored at 4℃. Morphological identification of the fungi The strain was cultured on PDA plates at 26℃ for 5 days. If we observed the colonial morphology, e.g., shape, size, color and odor, we could describe spore in great details, and the microscopic features were measured from strains grown on PDA plates 2 days. Polyunsaturated fatty acids analysis The fatty acid methyl esters (FAME) were prepared according to the method of Morrison and Smith (Morrison and Smith, 1964). Mycelia powder (150 mg) was disrupted by mortar and pestle, mixed with 5 mL aether:chloroform (1:1) and incubated at room temperature for 16h for the thorough extraction of cellular fatty acids. Then the extracts were subjected to saponification by adding 5 mL 5% KOH methanol solution. The FAME were subsequently analyzed by gas chromatography (GC; GC-2010, Shimadzu, Kyoto, Japan). A fused capillary column NUKOL 30 m × 0.25 mm Supelco was used in this research, at a column temperature of 170℃,
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an injection temperature of 250℃, and a detector temperature of 250℃. The mass spectrum of a new peak was compared with that of the standards (Sigma) for fatty acid identification. Molecular identifying of the Strain The mycelia were grown in 500 ml Erlenmeyer flasks containing 200 ml of potato dextrose broth with 100 rpm for 5 days at 26±1℃. The mycelia were harvested by filtration and grinded with liquid nitrogen thoroughly. Total genomic DNA was extracted by using the method suggested by the instructions in the Genomic Prep Fungal
DNA
midi
kit
(Omega
Bio-Tek,
Inc,
Norcross,
GA,
USA).
Primers
ITS1
(5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) was used for amplification and sequence of 18S rRNA gene. We used a polymerase chain reaction (PCR) protocol with a thermostable DNA polymerase (Takara, Japan) and cut out the amplified band about 600 bases in 1% TBE-agarose gel and then extracted it using Gene Clean Turbo (Qbiogene, Baton Rouge, LA, USA), and Sequenced in the ABI PRISM 77 DNA sequencer. The strain sequences of 18S rDNA were used as query sequence to search for similar sequences from GenBank and used for subsequent phylogenetic analyses. Cloning and Genetic engineering experiments of △6-desaturase gene 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 (Chomczynski and Sacchi 1987) based on guanidinium thiocyanate and stored at -80℃ for future use. The GeneRacer kit from Invitrogen (USA) was used for the cloning of full-length △6-desaturase cDNA of the
through
rapid
amplification
of
cDNA
ends
(5’-AAGGTGTACGATGTGACTGAATTCGT-3’)
(RACE).
A
pair
of
PCR
primers
and
FD6O RD6O
(5’-TGCTCGATTTGATAGTTCAATCCACC-3’), which corresponded to two conserved sites of fungal △ 6-desaturase genes, were designed and synthesized (Shanghai Sangon Biotechnology Corporation Ltd, Shanghai, China) to amplify the conserved region of the target gene. PCR amplification was performed according to the method of Lu et al (Lu et al., 2009). Sequence alignments, ORF translation and molecular mass calculation of predicted protein were carried out on Vector NTI Suite 9.0. GenBank BLASTs were done on NCBI (http://www.ncbi.nlm.nih.gov/), while structural analysis of predicted RoD6D protein was carried out on online softwares linked by websites (http:// www.expasy.org). The ORF fragment identified by sequencing was cut out from the recombinant pMD18-T using XhoI and
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HindIII double digestion and recovered to be ligated with the XhoI and HindIII digested yeast expression vector pYES2.0 (Invitrogen, USA) to generate the recombinant plasmid pYRoD6D. S. cerevisiae was transformed with pYRoD6D and pYES2.0 respectively by using the lithium acetate method (Lu et al., 2009). All experiments were conducted three times with three replicates of each isolate. The data are interpreted as mean±SD (n=9).
Results Isolation and Identification of the Gamma linolenic acid producing fungi In order to obtain high yield strain of GLA, the strain accumulating GLA was isolated by using Sudan black staining. Firstly, PDA plate was used for isolation of fungal, thirty strains in PDA plates were selected and then Sudan black was used to stain the strains. The darker strains were selected, indicating that the Strains for potential of GLA produced strains. The mycelia Fatty acids were transformed to methyl esters and analyzed by gas chromatography (GC) (Fig 1). GLA standards were used as reference for the retention times. According to these characteristics described above, strain DR3 was screening. The strain DR3 showed a content of GLA in total lipid of 16.68%, compared with another known as strains production of GLA we screened the strains DR3 with higher production capacity. (Table 1) The strains colonies reached 3–5 cm diameter on PDA after 5 days, color variable with state of sporulation grew from white to grayish and rhizoids were well developed. Sporangiospore arose from basal hyphae. According to these characteristics described above, strain DR3 was identified as one certain species of Rhizopus. Meanwhile, the 18S rDNA sequence of strain DR3 was also analyzed. In the NCBI GenBank under the accession numbers KC571633. The sequence with 623bp showed 100% identity with that of Rhizopus oryzae strain CNM-CM-6256(JX120700). In the light of the 18S rRNA genes sequences of strain DR3 and other 6 representative reference taxa, the Neighbor-joining tree revealed distinctly that fungus DR3 was as a member of the Rhizopus clade (Fig. 2). According to the phylogenetic tree based on related fungal, the strain DR3 was highly similar to Rhizopus oryzae, which was identical with the result of morphological identification Therefore, the strain DR3 was identified as Rhizopus oryzae. Cloning of the R. oryzae DR3 △6-fatty acid desaturase gene A cDNA fragment was proved to be of 881 bp in length by sequencing. NCBI Blastx search of this fragment showed broad homologies to △6-desaturases from fungi. The sequenced full-length cDNA of RoD6D (without poly A tail) was 1176bp long, which is completely in consensus with the putative full-length cDNA. The primer pair was also used to amplify the genomic DNA template of R. oryzae strain DR3 to characterize the This article is protected by copyright. All rights reserved.
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intron/exon structure information of this gene. It yielded a bright DNA band which was proved to be 1565bp 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 sequence completely coincided with the full-length cDNA of RoD6D, which suggested that we have successfully isolated both the mRNA and the genomic sequences of this gene. The mRNA and corresponding genomic nucleotide sequences of RoD6D were deposited in the NCBI GenBank under the accession numbers JQ063116 and JQ063117 respectively. Sequence comparison and structure prediction 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. A BLAST analysis using the D6DR amino acid sequence showed the similarity with other PUFA of the desaturase in the GenBank database. A multiple sequence alignment of this sequence against similar desaturase (Fig.3) the cytochrome b5 domain conserved sequence ‘‘HPGG’’ and the three histidine-box sequences ‘‘HDFGH’’, ‘‘HNTHH’’ and ‘‘QIEH’’ of the desaturase domain. On NCBI local alignment, RoD6D showed 63% identities and 64% positives to △ 6-desaturases from R. oryzae (AAS93682) and R.stolonifer(AAX22052), but less than 31% identities and 61% positives to all other desaturases. Phylogenetic analysis also demonstrated the homology between RoD6D and △6-desaturases from various organisms (Fig 4). Signal P V2.0 prediction did not detect any signal peptide sequence in RoD6D. TMHMM prediction yielded 4 potential transmembrane helices in RoD6D (Fig. 5). Confirmation of the activity of the △6-fatty acid desaturase 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 pYRoD6D, 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 pYRoD6D encoded a △-6 fatty acid desaturase. The expressed enzyme specifically converted the incorporated LA (0.4mM) to GLA (Fig.6). Comparison of different production methods Currently on the market GLA is commodity obtained from seeds of plant and microorganisms source. But, in Table 2, we can clearly see that fermentation Douchi production of GLA is easy, compared with other methods.
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Discussion Dietary polyunsaturated fatty acids are indispensable for human cells to maintain their structure and function (Calder and Newsholme, 1992). GLA is a PUFA with many medicinal and healthy promoting effects, such as kill cancer cell and anti-infection (Gill and Valivety, 1997; Raclot et al., 1997). The main sources of GLA are plant seeds such as black currant and fungi from fermentation, like species of Mortierella, and Mucor are known (Kapoor and Huang, 2006; Kenny et al., 2000; Sakuradani et al., 2009). However, it is obvious that GLA production from the current sources is inadequate for supplying the expanding market due to such significant problems as low productivity, insecurity, expensive downstream processing and etc (Wan et al., 2009). So, this is still the hot spot of to find more excellent GLA producing strain. Many species of the GLA producing fungal have been isolated, such as Mortierella alpine, Rhizopus stolonifer, Mucor. However, it has fewer reports that R. oryzae has not been used as one kind of producin GLA fungal in the Douchi. In order to obtain high yield strain GLA with Sultan black staining is a good screening method. Strain DR3, which is able to produce abundant GLA, was isolated from the Douchi by using Sudan black staining. For further research of this strain DR3 was identified. Morphological identification which employed light microscope showed that DR3 was Rhizopus sp.18S rDNA gene were then cloned sequences proved that strain DR3 was R. oryzae. In this study, the R. oryzae in spontaneously fermented Douchi was studied by conventional culture-dependent methods combined with molecular biological methods.
Although the GLA can be got in different biosynthesis pathways of strains (Laho et al., 2011; Certik et al., 2006; Zhang et al., 2004), and △6-fatty acid desaturase genes has been previously cloned and characterized from several fungi, such as Rhizopus nigricans, R.arrhizus (McCue and Shetty,2003; Fujita et al.,2001; Fujita et al.,2003). We have cloned and confirmed of the activity a new △6-fatty acid desaturase gene involved in GLA biosynthesis in strain DR3. This enzyme contains the signature desaturase motif four membrane domains, and share extensive similarity in secondary structure arrangement. It suggests that they retained the correct conformation and intracellular location. However, they share less identity to other △6-fatty acid desaturase such as the △6-fatty acid desaturase from R. oryzae AAS93682 (63 % identity). The percentage of GLA was 16.68% of the total fatty acids. This result showed that some special characterization of △6-fatty acid desaturase of R. oryzae DR3. It also shows that △6-fatty acid desaturase activity may be enhanced by various effects, For example, the codon optimization or the length of sequence to further improve the level of expression obtained (Xue et al., 2013; Lu et al., 2013; Song et al., 2013). However, fungal sources would bring about new issues of GLA safety. So, how to reduce the risk of food pollution is important, and direct consumption of This article is protected by copyright. All rights reserved.
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natural foods containing GLA will best meet the needs of people. Nowadays, much attention has been paid by consumers towards natural bioactive compounds as functional ingredients in the diet. Although the GLA can be got in different biosynthesis pathways and strains (Laho et al., 2011; Certik et al., 2006; Zhang et al., 2004), but direct consumption of natural foods containing GLA will best meet the needs of people. With the growing interest in traditional soybean foods, the functional ingredients (e.g., isoflavone, aqueous extract, and aroma components) contained in Douchi has been fully investigated (Hesseltine and Wang 1972; Chaiyasut et al., 2010). The recent study indicates that Douchi is useful to relieve tiredness, weakness, insomnia, and poor appetite (Zhang et al 2004), and is able to prevent and cure diabetic (Chen et al., 2007; McCue et al., 2003; Fujita et al., 2001). However, preliminary experiment of GLA in commercial Douchi from local supermarkets sample did not contain GLA. So, we isolated strain DR3 of R. oryzae from Douchi. According to the phylogenetic tree based on related fungal, the strain DR3 was identified as R. oryzae. By using GC analysis we have discovered that this species also synthesizes PUFA, especially GLA. R. oryzae is used in the food industry and its products are generally recognised as safety. Therefore gene isolation and functional characterization of △6-fatty acid desaturases from R. oryzae are a prerequisite for the elucidation of the related molecular mechanism and the development of applicable products. The bioactivity of the PUFA produced by strain DR3, R. oryzae, will be further studied and the results will be published in the future. This study has also laid a foundation for further research on developing functional Douchi products. It also shows that Douchi contains a large natural diverse composition and some strains could be selected as starters for functional fermented foods. Confirmed This research was supported by the Key Project of Chinese Ministry of Education（200202128）, China National “948” Program（2003Q04） Acknowledgements This research was supported by the Key Project of Chinese Ministry of Education（200202128）, China National “948” Program（2003Q04）.
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Fig. 1 Fatty acid composition of R. oryzae DR3 by GC analysis (Red line is standards of GLA)
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Fig. 2 Neighbor-joining tree of Fungal R. oryzae DR3 (KC571633) based on 18S rRNA gene sequences. Confidence values above 100% obtained from a 1000-replicate bootstrap analysis are shown at the branch nodes. Bootstrap values from neighbor-joining method were determined. Mucor fragilis (JN198474) and Mucor plumbeus strain UASWS0823 (JX139728) were used as the out group.
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Fig. 3 Sequence alignment of deduced amino acids of delta 6-desaturase from fungus R. oryzae DR3 (ROD6) with those of R. delemar (RDD6), R. stolonifer (RSD6), Mortierella alpine (M. alpine ). A cytochrom b5-like domain and three conserved histidine-rich motifs are underlined.
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Fig. 4 A Phylogenetic relationship between RoD6D and △6-desaturases from various fungi. ADD50000 Sparus aurata, AAC49700 Borago officinalis. ADI49410 Phaeodactylum tricornutum, ADE06661 Mortierella alpina, AAX22052 Rhizopus stolonifer, ADX21039 Mucor circinelloides f. lusitanicus, AAS93682 Rhizopus oryzae. ADX21038 Mucor fuscus. AFD18733 Rhizopus oryzae DR3
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Fig. 5 Hydropathy profile of RoD6D of R.oryzae DR3
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Fig. 6 Identification of GLA in transgenic S. cerevisiae by GC analysis A: S. cerevisiae transformed with control vector pYES2.0. B: S. cerevisiae transformed with the recombinant plasmid pYRoD6D. The arrowhead indicates the novel peak of GLA
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Table:1 Relative percentage of fatty acid and GLA of fungal Strain
Total fatty acids
GLA (% of total fatty acids)
Cunninghamella echinulata
36.73
6.4
Mucor rouxii
31.25
15.42
Rhizopus nigricans
54.83
24.32
DR3
41.25
16.68
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Table:2
Compared with other methods
Sample
New technology Douchi
Supermarket Douchi
Fungal lipid
GLA in total fatty acids
3.54±0.36
0.00
16.68
Food
yes
yes
No
Purification
No need
No need
Need
(%)
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