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Transformation of oil-producing fungus, Mortierella alpina 1S-4, using Zeocin, and application to arachidonic acid production
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 100, No. 6, 617–622. 2005 DOI: 10.1263/jbb.100.617
© 2005, The Society for Biotechnology, Japan
Transformation of Oil-Producing Fungus, Mortierella alpina 1S-4, Using Zeocin, and Application to Arachidonic Acid Production Seiki Takeno,1 Eiji Sakuradani,1 Akiko Tomi,1 Misa Inohara-Ochiai,2 Hiroshi Kawashima,3 and Sakayu Shimizu1* Laboratory of Fermentation Physiology and Applied Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan,1 Institute for Advanced Technology, Suntory Ltd., 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan,2 and Institute for Health Care Science, Suntory Ltd., 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan3 Received 13 July 2005/Accepted 16 August 2005
The arachidonic acid-producing fungus Mortierella alpina 1S-4, an industrial strain, was endowed with Zeocin resistance by integration of the Zeocin-resistance gene at the rDNA locus of genomic DNA. Plasmid DNA was introduced into spores by microprojectile bombardment. Twenty mg/ml Zeocin completely inhibited the germination of M. alpina 1S-4 spores, and decreased the growth rate of fungal filaments to some extent. It was suggested that preincubation period and temperature had a great influence on transformation efficiency. Four out of 26 isolated transformants were selected. Molecular analysis of these stable transformants showed that the plasmid DNA was integrated into the rDNA locus of the genomic DNA. We expect that this system will be applied for useful oil production by gene manipulation of M. alpina 1S-4 and its derivative mutants. On the basis of the fundamental transformation system, we also tried to overexpress a homologous polyunsaturated fatty acid elongase gene, which has been reported to be included in the rate-limiting step for arachidonic acid production, thereby leading to increased arachidonic acid production. [Key words: Mortierella alpina 1S-4, transformation, Zeocin, Zeocin resistance, stable transformant]
A filamentous fungus, Mortierella alpina 1S-4, was found to be a potent producer of polyunsaturated fatty acid (PUFA)-containing lipids in this laboratory (1). This strain is unique in that it produces some C-20 PUFAs, e.g., dihomo-γ-linolenic acid (DGLA, 20:3n-6), arachidonic acid (AA, 20:4n-6), and eicosapentaenoic acid (EPA, 20:5n-3), that play important roles as precursors of eicosanoids of signaling molecules. We have studied the fatty acid metabolism in this strain and have succeeded in applying into the industrial production of AA (2). Therefore, this fungus is a good model for analyzing a fatty acid desaturation and/or elongation system from both the fundamental and applied standpoints. In addition, we have isolated a number of derivative mutants of this fungus, and their fatty acid synthetic pathways have been determined through analysis of fatty acid composition or accumulation (3–5). Wynn and coworkers, have deduced the rate-limiting step of AA production and the NADPH-producing step responsible for fatty acid synthesis by enzymatic analysis (6, 7): the rate-limiting step for AA production is the elongation of γ-linolenic acid (GLA, 18:3n-6) to DGLA. The elongase responsible for the conversion of GLA to DGLA is designated as the GLELO protein. We proved the hypothesis through molecular ana-
lyses (8). We have cloned and sequenced the fatty acid desaturase and elongase genes (9–11). This achievement should be useful for future studies and for the oil industry. Therefore, the establishment of a transformation system for this fungus has become urgently required. We have established a transformation system for the fungus and proposed its application to the oil industry (12). However, the work involved the use of a uracil auxotrophic mutant (ura5– strain) of M. alpina 1S-4 as the host and the homologous ura5 gene as the selective marker. We also increased AA productivity through the overexpression of the GLELO gene in the ura5– strain (8). However, AA productivity obtained through the previous transformation system was thought to be enhanced using the wild-type strain as the host instead of the ura5– strain; the ura5– strain was not suitable for the application because of its low growth rate and lipid productivity. In spite of the expectations, the former system cannot be directly applied to wild-type M. alpina 1S-4 or its derivative mutants. Hence, in the present study we tried to develop another method, particularly, one involving antibiotic markers. Drug resistance markers have proved to be of great use in filamentous fungi (13–15). One disadvantage, however, is that the resistance allele may not show a significant dominance over the wild-type allele, resulting in selection difficulties. A minor disadvantage of many of these selective markers is that compounds used to select for
* Corresponding author. e-mail: [email protected] phone: +81-(0)75-753-6115 fax: +81-(0)75-753-6128 617
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transformants are often very expensive. In this study, we optimized the conditions for the transformation of M. alpina 1S-4 by microprojectile bombardment and succeeded in obtaining transformants resistant to an antibiotic, Zeocin (Invitrogen, Carlsbad, CA, USA). Moreover, we tried to overexpress a homologous fatty acid elongase (GLELOp) gene in the wild-type strain to enhance AA productivity. MATERIALS AND METHODS Enzymes and chemicals Restriction enzymes and other DNA-modifying enzymes were obtained from Takara Bio (Shiga) and New England BioLabs (Beverly, MA, USA). Zeocin was purchased from Invitrogen. All other chemicals were of the highest purity commercially available. Strains, media, and growth conditions M. alpina 1S-4 (AKU 3998), deposited in Kyoto University, was used for transformation. The GY medium (pH 6.5) containing 2% glucose and 1% Bacto yeast extract (Becton, Dickinson and Company, Sparks, MD, USA) was used for routine cultivation and as a selective medium by the addition of antibiotics. To collect M. alpina 1S-4 spores, the Czapek-Dox medium was used as described previously (16). All fungal cultivations were performed at 28°C. Escherichia coli DH5α was used for DNA manipulation and cultivated at 37°C under vigorous shaking (300 rpm). Construction of transformation vector, pDZeo and pDZeoGLELO Vector pDura5 (12) was previously constructed by the modification of vector pD4, which had been used for the transformation of M. alpina CBS 224.37, and which was kindly supplied by Dr. Archer, D.B. (University of Nottingham, UK). The PCR amplification of a modified Zeocin-resistance gene was carried out in a total volume of 30 µl containing 200 ng of pEM7/Zeo (Invitrogen), 0.2 µl of LA Taq (Takara Bio), 10 ×LA Taq buffer, 2 mM MgCl2, 10 pmol of primers and 200 µM of each dNTP. Two primers, ZeoFNcoI (5′-AAACCATGGCCAAGTTGACCAGT-3′) and ZeoRxEoB (5′-AGAGGATCCCCGGGAATTATCAGT-3′), were designed on the basis of the Zeocin resistance gene of pEM7/Zeo and had annealing temperatures of being 61.6°C. These two primers contained a NcoI restriction site and a BamHI restriction site, respectively (underlined). The resulting 400-bp fragment was cloned into the pT7 Blue T-vector (Novagen, Madison, WI, USA), the resulting vector was designated as pT7-Zeo, and the sequence was confirmed. The modified Zeocin-resistance gene cut out from pT7-Zeo with NcoI and BamHI was ligated to pDura5 (without the ura5 gene) digested with NcoI and BamHI to construct pDZeo for the transformation of M. alpina by microprojectile bombardment (Fig. 1). The transformation vector pDZeoGLELO was constructed so that the expression system of a homologous GLELO gene (AB193123) was integrated into pDZeo. The expression unit was constructed as described previously (8). The expression unit was digested with EcoRI and ligated to pDZeo digested with the same enzyme to generate pDZeoGLELO (Fig. 1). Transformation of M. alpina with pDZeo and pDZeoGLELO by microprojectile bombardment A PDS-1000/He particle delivery system (Bio-Rad Laboratories, Hercules, CA, USA) was used for the transformation under the same conditions as described previously (12). In the case of transformation with pDZeo, 4.0 ×106 spores were spread on a plate containing the GY medium without Zeocin. After the bombardment, the plate was statically incubated at 12°C or 28°C for 0–15 h. Swollen spores, just prior to germination, were collected by suspending them in ~500 µl of Tween 80 solution (0.3 µl/ml of distilled H2O), and then spread on the selective GY medium containing 20 mg/ml Zeocin, followed by incubation at
FIG. 1. Maps of M. alpina 1S-4 transformation vector pDZeo and pDZeoGLELO. The details of the genes and the method used to construct the vector are given in Materials and Methods. Zeo, Zeocin resistance gene; bla, ampicillin resistance gene; his H4.1p, M. alpina histone H4.1 promoter fragment from strain CBS 528.72; trpCt, Aspergillus nidulans N-(5′-phosphoribosyl)anthranilate isomerase (trpC) transcription terminator; rDNA, M. alpina 1S-4 18S rDNA fragment. Restriction sites: E, EcoRI; N, NcoI; B, BamHI; X, XbaI; H, HindIII; S, SspI.
28°C. After 2 d, transformants resistant to Zeocin were isolated and maintained in the same medium as that for cultivation under selective conditions for further analyses. To select stable transformants, the following method was adopted: Individual mycelia were inoculated onto the GY medium without Zeocin. After 2 d, a mycelium was picked up from the outer part of a colony and then inoculated onto a fresh GY medium. The selection was repeated three times. Mycelia from the third culture were finally inoculated onto the GY medium containing 20 mg/ml Zeocin. Transformants grown on the selection medium were defined as being stable with Zeocin-resistance. In the case of transformation with pDZeoGLELO, intact spores of M. alpina 1S-4 wild-type strain were harvested from the surface of the Czapek–Dox agar medium (2.0×108 spores/300 cm2). Spores (4.0 ×107) were spread on a GY medium plate without Zeocin. Each plate was bombarded twice with a PDS-1000/He particle
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delivery system. After the bombardment, the plate was statically incubated at 12°C for 8–25.5 h. Swollen spores were collected by suspending them in ~500 ml of distilled H2O, spread onto the selective GY medium containing 20 mg/ml Zeocin, and then incubated at 28°C. After 2 d, mycelia were isolated and maintained in the same medium under selective conditions for further analyses. Stable transformants were selected by the same methods as in the case of pDZeo transformants. Confirmation of transformation by PCR Genomic DNA of transformants was obtained from mycelia grown in the GY medium. Genomic DNA of M. alpina strains was prepared according to a method described previously (12). Transformants were confirmed by PCR using the forward primer ZeoFNcoI and the reverse primer ZeoRxEoB. The integration of plasmid pDZeo or pDZeoGLELO into an appropriate chromosomal rDNA region was verified using the vector-specific forward primer RDNA1 (5′-ACAGGTACACTTGTTTAGAG-3′) and the reverse primer RDNA2 (5′-CGCTGCGTTCTTCATCGATG-3′), both of which were designed with reference to the report of Mackenzie et al. (17). RDNA1 and RDNA2 respectively anneal immediately upstream of the XbaI site in the trpC terminator region and the 5.8S rDNA gene; this gene lies downstream of the 18S rDNA region in the genomic DNA but not in these vectors. In the case of pDZeoGLELO transformants, the forward primer GLELOF (5′-CACCATGGAGTCGATTGCGC-3′) and the reverse primer GLELOR (5′-GTGGATCCTTACTGCAACTTCCTTGCCTT-3′) were also available to detect GLELO cDNA in the genomic DNA (8). Fatty acid analyses The spores of M. alpina recombinant cells transformed with pDZeoGLELO and those of wild-type cells (host cells) were inoculated into a 20-ml Erlenmeyer flask containing 5 ml of GY medium consisting of 2% glucose and 1% yeast extract. The culturing was carried out at 28°C with reciprocal shaking (120 strokes/min) for 10 d. Fatty acid analysis was performed basically as described in a previous paper (9).
RESULTS Screening of antibiotics that inhibit growth of M. alpina 1S-4 The first step was to screen for useful antibiotics that inhibit the growth of M. alpina 1S-4. Growth experiments were carried out on the GY agar medium containing various concentrations of antibiotics. As a result, Zeocin was considered to be a possible candidate (data not shown): other antibiotics, such as blasticidin S, bleomycin, Geneticin, hygromycin B, and oligomycin did not inhibit the growth of the strain, whereas a high concentration of Zeocin (20 mg/ml) did, although not completely. However, the germination of spores was completely inhibited by Zeocin. Since the transformation system used in this study involved gene transfer into spores, Zeocin was considered suitable. The concentration of Zeocin was fixed at 20 mg/ml unless otherwise stated. Transformation of M. alpina 1S-4 and optimization of conditions The M. alpina 1S-4 transformation vector pDZeo was developed by means of the methods as described in Materials and Methods. pDZeo was designed to be integrated into the rDNA locus of M. alpina 1S-4 genomic DNA. When pDZeo was bombarded into the spores on the GY medium containing Zeocin (selective medium), mycelia did not appear, suggesting that the integration of pDZeo into the genomic DNA and the expression of the Zeocin-resistance gene must have occurred in the host cells
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TABLE 1. Effects of preincubation temperature and period on transformation frequency prior to transfer to selective medium Preincubation Number of transforTemp. Period Morphological appearance of spores mantsa (°C) (h) 12 0 Small and rod-shaped, before germination –b 6 Small and rod-shaped, before germination 9 10 Swollen and spherical, before germination 14 15 ~50% of spores germinated 10 21 All spores germinated N.D.c 28 0 Small and rod-shaped, before germination – 6 Swollen and spherical, before germination 12 10–21 All spores germinated N.D. Bombarded spores were grown on the GY medium without Zeocin at 12°C or 28°C for the designated period (preincubation). After the cultivation, spores were collected and transferred to the Zeocin-containing selective medium. Morphological appearances of spores just prior to the transfer were observed under a microscope. a The number of transformants was determined by counting colonies on the Zeocin-containing selective medium. b –, Not detected. c N.D., Not determined.
before they were placed on the Zeocin-containing medium. Therefore, once gene transfer into the spores had occurred on the GY medium, and cultivation at 12°C for 2 d to allow germination and some growth had been performed, the GY medium containing Zeocin was poured onto each plate. As a result, the surfaces of the plates were covered with numerous mycelia that could not be selected. On the basis of the finding that spores never grew on Zeocin-containing medium and the mycelia were hardly affected by Zeocin, the following method was examined: Vector-bombarded spores on the GY medium were preincubated just prior to germination, and then the spores were collected from the surface of each plate, and spread onto the selective medium, and then cultivated at 28°C. As a result, many transformants were isolated. We considered that preincubation must be the most important step, and thus optimized preincubation temperature and period (Table 1). The highest transformation frequency was obtained when the preincubation was at 12°C for 10 h, and spores were fully swollen just prior to germination in this state. At 12°C for 21 h or at 28°C for 10–21 h, all the spores completely germinated and stuck to the surface of the medium. Up to seven strains were randomly isolated from each selective medium, and the strains were characterized by growth and genetic analyses. Analyses of stability of transformants and comfirmation of transformation by PCR Stable transformants were selected as described in Materials and Methods. Out of 26 transformants obtained, regardless of the preincubation conditions, 15 were judged to be stable. The appearance rate of stable transformants reached about 60% regardless of the conditions. Secondly, of the 15 stable transformants, four (#1, 6, 13, and 28) were selected for further analyses, because all four could grow vigorously under the Zeocincontaining conditions (up to 20 mg/ml) (Fig. 2). Genomic DNA was prepared from each transformant cultivated in the GY liquid medium. The strategy for transformation with pDZeo was based on homologous integration into rDNA. Transformed cells, therefore, were confirmed by PCR using the primers RDNA1 and RDNA2. A band corre-
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FIG. 2. Growth of wild-type strain and transformants on GY medium with or without Zeocin. Each mycelium was inoculated onto the GY medium with or without Zeocin, and then incubated at 28°C for 4 d.
sponding to a 1.5-kb amplified fragment appeared on an agarose gel only when pDZeo was integrated into the chromosomal rDNA locus. For further confirmation, the vectorspecific reverse primer ZeoRxEoB was also used with the forward primer ZeoFNcoI to confirm the presence of pDZeo in transformants. As shown in Fig. 3, the 1.5-kb amplified fragment obtained with RDNA1 and RDNA2 indicated that pDZeo existed at the rDNA locus of the genomic DNAs of the transformants. On the other hand, a nonspecific band corresponding to a 2.8-kb fragment was observed for all transformants and host cells. This nonspecific band was determined to represent a certain rDNA region amplified with these two primers: primer RDNA1 anneals at this unknown region of 18S rDNA, leading to the generation of this nonspecific band (data not shown). In the cases of #6 and #28, the intensity of the nonspecific bands was stronger than that of bands corresponding to 1.5-kb fragments. This tendency has frequently been observed in unstable transformants (data not shown). Transformation of M. alpina 1S-4 with pDZeoGLELO We reported that GLELO gene overexpression leads to a high productivity of AA in the ura5– strain (8). In the present study, we tried to overexpress the gene in wild-type 1S-4. Wild-type M. alpina with pDZeoGLELO was transformed as described in Materials and Methods. Out of 60 transformants obtained, two stable transformants (#55 and #63) were selected. The transformation frequency was 6.8 transformants/µg of transformation vector. Both transformants could grow on the GY medium containing 20 mg/ml Zeocin more vigorously than the wild-type strain (data not shown). The transformation was confirmed by PCR (data
FIG. 3. Confirmation of transformation by PCR. Transformation was confirmed by PCR using two primers, ZeoFNcoI/ZeoRxEoB (top) and RDNA1/RDNA2 (bottom). M denotes 2-log DNA ladder markers (New England BioLabs).
not shown). In addition, real-time quantitative PCR analysis showed that the quantity of GLELO RNA in transformant #63 was 3.4-fold higher than that in the host strain (wildtype strain) on the 4th day (data not shown). To compare fatty acid productivities, these two transformants were grown in the GY medium at 28°C for 10 d and fatty acid analyses were carried out (Table 2). Marked differences in growth were not observed. However, the content of AA in the pDZeoGLELO transformants was higher than that in the host strain and pDZeo transformants. GLA content in the pDZeoGLELO transformants decreased concomitantly with the increase in AA content. DISCUSSION In our previous study (12), we established a basic transformation system for M. alpina using its uracil auxotrophic mutants. The system could only be applied to uracil auxotrophs. We then attempted to construct a universal system
TABLE 2. Comparison of PUFA productivities of M. alpina 1S-4, pDZeo transformant, and two pDZeoGLELO transformants Growtha Total FAb (mg/ml) (mg/ml)
FA composition (wt%) 16:0 18:0 18:1n-9 18:2n-6 GLA DGLA AA Others 9.18±0.02 4.70±0.47 10.4±0.21 3.34±0.24 14.3±0.10 6.73±0.27 5.09±0.07 3.94±0.09 53.4±0.98 2.88±0.01 Wild-type 1S-4 pDZeo transformant #1 9.66±0.08 6.18±0.10 10.4±0.28 3.99±0.15 10.1±0.25 6.61± 0.11 4.32±0.00 3.99±0.09 58.2±0.86 2.39±0.04 pDZeoGLELO transformant #55 9.91±0.11 6.88±0.30 9.96±0.33 3.99±0.53 8.24±0.48 5.5±0.06 4.02±0.22 3.93±0.11 62.2±1.12 2.18±0.07 pDZeoGLELO transformant #63 9.72±0.02 7.09±0.95 8.29±0.21 2.37±0.28 6.12±0.32 4.12±0.04 3.41±0.05 3.23±0.40 71.2±1.34 1.31±0.27 All strains were grown at 28°C for 10 d in 5 ml of the GY medium under reciprocal shaking (120 strokes/min). These values are the means of duplicate experiments. a Milligram of dry cell weight/ml of culture broth. b Milligram of total fatty acid/ml of culture broth. FA, Fatty acid; 16:0, palmitic acid; 18:0, stearic acid; 18:1n-9, oleic acid; 18:2n-6, linoleic acid; GLA, γ-linolenic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid. Strains
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for the wild-type strain, M. alpina 1S-4, and various mutants, which had been isolated, characterized and utilized for PUFA production, and for the study of PUFA metabolism. Antibiotics generally show a strong inhibitory effect on prokaryote growth, but not always on eukaryotes such as fungi. Mackenzie et al. reported transformation systems using M. alpina CBS 224.37, which is sensitive to hygromycin B (17). Although the morphological shape of M. alpina 1S-4 is very similar to that of M. alpina CBS 224.37, M. alpina 1S-4 is not sensitive to hygromycin B even at high concentrations. Out of some antibiotics reported to be effective against filamentous fungi, we found that Zeocin completely inhibited the germination of spores of M. alpina 1S-4. In this study, we developed another transformation method using Zeocin as a selectable marker, which might be useful in the development of transformation systems for other organisms. We consider that the selection of stable transformants is the most important step for further analysis and practical studies. From the application viewpoint, PUFA production is performed in submerged cultures and the GY medium has been reported to be the most suitable medium for this (18, 19). The cultivation of unstable transformants in a Zeocincontaining medium to generate selective pressure leads to high costs and biohazard problems, but stable transformants require no continuous selective pressure during cultivation. In the previous transformation method using uracil auxotrophs on a uracil-free synthetic medium, the appearance rate of stable transformants was about 10% for all isolates. On the other hand, we obtained them at a rate of 60% in this study. The lower rate for uracil auxotroph transformation may be attributed to the random mutation occurring by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment. Consequently, we obtained stable transformants resistant to Zeocin at a higher rate. We analyzed differences in genetic property and growth among four stable transformants (#1, 6, 13, and 28). They could grow on the GY medium containing 20 mg/ml Zeocin after serial cultivation on the GY medium. Their growth rate was determined to be the same as that of the wild-type strain in the absence of Zeocin (Fig. 2). This observation is suitable for practical applications, as mentioned above. In addition, genetic analyses revealed some differences among the transformants (Fig. 3). Two PCR products, 1.5-kb and 2.8-kb fragments, were detected for all samples when the amplification was performed using RDNA1 and RDNA2. The 2.8-kb fragment was identified as a certain region in the rDNA locus (data not shown). The ratio of the amounts of the fragments differed among individual transformants. This difference cannot be explained but the ratio affected the degree of stability, that is, transformants with higher amounts of the 1.5-kb fragment were defined as stable. Therefore, #1 and #13 were defined as more stable transformants than #6 and #28. The copy number of the transformation vector in the transformants was not determined, but it is clear that #1 and #13 contain more GLELO genes in their genomic DNA than #6 and #28 as shown in Fig. 3. We investigated the fatty acid compositions of pDZeo and pDZeoGLELO transformants (Table 2). pDZeo trans-
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formant #1, as the control strain, showed the same rate of growth as the wild-type 1S-4. Therefore, it is considered that the expression of the marker gene does not affect growth. On the other hand, the total fatty acid content of the pDZeo transformant was higher than that of the wild-type 1S-4. The reason remains unclear, but one hypothesis can be proposed. Wynn et al. reported that the exhaustion of the nitrogen source triggers lipid accumulation (20). Overproduction of a Zeocin-resistant protein may accelerate the exhaustion of the nitrogen source, leading to a higher lipid accumulation. However, the control strain showed a fatty acid composition comparable to that of the host cells. The results provide important information for further study. The fatty acid compositions of two GLELO-overexpressing transformants were different compared with those of the host cells and pDZeo transformants. In the case of pDZeoGLELO transformant #63, the AA composition was 1.2-fold higher than that of the pDZeo transformants. In addition, the compositions of other fatty acids generally decreased, which suggests a prompt fatty acid modification to AA. We previously obtained an equal increase in the rate of AA production using ura5– strain as the host (8). However, the productivity of pDZeoGLELO transformant #63 has never been obtained. The present technique led to an increase in AA productivity of the wild-type strain (from 2.51 to 5.05 mg of AA/ml of culture broth). The increased productivity is thought to be derived from the overexpression of the GLELO gene. The previous system using ura5– strains as the host (8) did not realize such a high productivity of AA; thus the present system is useful for increasing the fatty acid production. In this study, we succeeded in establishing of an overall transformation system for this fungus, and in improving the fatty acid composition. It is expected that genetic analyses and approaches will clarify the biosynthetic pathway of AA and contribute to the development of the oil industry. We aim to overexpress or destroy a gene involved in PUFA biosynthesis for the control of PUFA metabolism. ACKNOWLEDGMENTS We wish to thank Dr. David B. Archer (School of Life and Environmental Sciences, University of Nottingham, United Kingdom) for providing the Mortierella transformation vector. This work was supported in part by the New Energy and Industrial Technology Development Organization (NEDO), and a Grant-in-Aid for Scientific Research (no. 15658024 for S.S.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Biotechnol., 65, 419–425 (2004). 13. Lorito, M., Hayes, C. K., Di Pietro, A., and Harman, G. E.: Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA. Curr. Genet., 24, 349–356 (1993). 14. Te’o, V. S. J., Bergquist, P. L., and Nevalainen, K. M. H.: Biolistic transformation of Trichoderma reesei using the Bio-Rad seven barrels Hepta Adaptor system. J. Microbiol. Methods, 51, 393–399 (2002). 15. Dev, K. and Maheshwari, R.: Silencing of hygromycin phosphotransferase (hph) gene during sexual cycle and its reversible inactivation in heterokaryon of Neurospora crassa. Curr. Microbiol., 47, 220–225 (2003). 16. Takeno, S., Sakuradani, E., Murata, S., Inohara-Ochiai, M., Kawashima, H., Ashikari, T., and Shimizu, S.: Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4. Biosci. Biotechnol. Biochem., 68, 277–285 (2004). 17. Mackenzie, D. A., Wongwathanarat, P., Carter, A. T., and Archer, D. B.: Isolation and use of a homologous histone H4 promoter and a ribosomal DNA region in a transformation vector for the oil-producing fungus Mortierella alpina. Appl. Environ. Microbiol., 66, 4655–4661 (2000). 18. Shinmen, Y., Shimizu, S., Akimoto, K., Kawashima, H., and Yamada, H.: Production of arachidonic acid by Mortierella fungi. Selection of a potent producer and optimization of culture conditions for large-scale production. Appl. Microbiol. Biotechnol., 31, 11–16 (1989). 19. Kawashima, H., Nishihara, M., Hirano, Y., Kamada, N., Akimoto, K., Konishi, K., and Shimizu, S.: Production of 5,8,11-eicosatrienoic acid (Mead acid) by ∆6 desaturation activity-enhanced mutant derived from a ∆12 desaturase-defective mutant of an arachidonic acid-producing fungus, Mortierella alpina 1S-4. Appl. Environ. Microbiol., 63, 1820– 1825 (1997). 20. Wynn, J. P., Hamid, A. A., Li, Y., and Ratledge, C.: Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloids and Mortierella alpina. Microbiology, 147, 2857–2864 (2001).
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Transformation of Oil-Producing Fungus, Mortierella alpina 1S-4, Using Zeocin, and Application to Arachidonic Acid Production Seiki Takeno,1 Eiji Sakuradani,1 Akiko Tomi,1 Misa Inohara-Ochiai,2 Hiroshi Kawashima,3 and Sakayu Shimizu1* Laboratory of Fermentation Physiology and Applied Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan,1 Institute for Advanced Technology, Suntory Ltd., 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan,2 and Institute for Health Care Science, Suntory Ltd., 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan3 Received 13 July 2005/Accepted 16 August 2005
The arachidonic acid-producing fungus Mortierella alpina 1S-4, an industrial strain, was endowed with Zeocin resistance by integration of the Zeocin-resistance gene at the rDNA locus of genomic DNA. Plasmid DNA was introduced into spores by microprojectile bombardment. Twenty mg/ml Zeocin completely inhibited the germination of M. alpina 1S-4 spores, and decreased the growth rate of fungal filaments to some extent. It was suggested that preincubation period and temperature had a great influence on transformation efficiency. Four out of 26 isolated transformants were selected. Molecular analysis of these stable transformants showed that the plasmid DNA was integrated into the rDNA locus of the genomic DNA. We expect that this system will be applied for useful oil production by gene manipulation of M. alpina 1S-4 and its derivative mutants. On the basis of the fundamental transformation system, we also tried to overexpress a homologous polyunsaturated fatty acid elongase gene, which has been reported to be included in the rate-limiting step for arachidonic acid production, thereby leading to increased arachidonic acid production. [Key words: Mortierella alpina 1S-4, transformation, Zeocin, Zeocin resistance, stable transformant]
A filamentous fungus, Mortierella alpina 1S-4, was found to be a potent producer of polyunsaturated fatty acid (PUFA)-containing lipids in this laboratory (1). This strain is unique in that it produces some C-20 PUFAs, e.g., dihomo-γ-linolenic acid (DGLA, 20:3n-6), arachidonic acid (AA, 20:4n-6), and eicosapentaenoic acid (EPA, 20:5n-3), that play important roles as precursors of eicosanoids of signaling molecules. We have studied the fatty acid metabolism in this strain and have succeeded in applying into the industrial production of AA (2). Therefore, this fungus is a good model for analyzing a fatty acid desaturation and/or elongation system from both the fundamental and applied standpoints. In addition, we have isolated a number of derivative mutants of this fungus, and their fatty acid synthetic pathways have been determined through analysis of fatty acid composition or accumulation (3–5). Wynn and coworkers, have deduced the rate-limiting step of AA production and the NADPH-producing step responsible for fatty acid synthesis by enzymatic analysis (6, 7): the rate-limiting step for AA production is the elongation of γ-linolenic acid (GLA, 18:3n-6) to DGLA. The elongase responsible for the conversion of GLA to DGLA is designated as the GLELO protein. We proved the hypothesis through molecular ana-
lyses (8). We have cloned and sequenced the fatty acid desaturase and elongase genes (9–11). This achievement should be useful for future studies and for the oil industry. Therefore, the establishment of a transformation system for this fungus has become urgently required. We have established a transformation system for the fungus and proposed its application to the oil industry (12). However, the work involved the use of a uracil auxotrophic mutant (ura5– strain) of M. alpina 1S-4 as the host and the homologous ura5 gene as the selective marker. We also increased AA productivity through the overexpression of the GLELO gene in the ura5– strain (8). However, AA productivity obtained through the previous transformation system was thought to be enhanced using the wild-type strain as the host instead of the ura5– strain; the ura5– strain was not suitable for the application because of its low growth rate and lipid productivity. In spite of the expectations, the former system cannot be directly applied to wild-type M. alpina 1S-4 or its derivative mutants. Hence, in the present study we tried to develop another method, particularly, one involving antibiotic markers. Drug resistance markers have proved to be of great use in filamentous fungi (13–15). One disadvantage, however, is that the resistance allele may not show a significant dominance over the wild-type allele, resulting in selection difficulties. A minor disadvantage of many of these selective markers is that compounds used to select for
* Corresponding author. e-mail: [email protected] phone: +81-(0)75-753-6115 fax: +81-(0)75-753-6128 617
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transformants are often very expensive. In this study, we optimized the conditions for the transformation of M. alpina 1S-4 by microprojectile bombardment and succeeded in obtaining transformants resistant to an antibiotic, Zeocin (Invitrogen, Carlsbad, CA, USA). Moreover, we tried to overexpress a homologous fatty acid elongase (GLELOp) gene in the wild-type strain to enhance AA productivity. MATERIALS AND METHODS Enzymes and chemicals Restriction enzymes and other DNA-modifying enzymes were obtained from Takara Bio (Shiga) and New England BioLabs (Beverly, MA, USA). Zeocin was purchased from Invitrogen. All other chemicals were of the highest purity commercially available. Strains, media, and growth conditions M. alpina 1S-4 (AKU 3998), deposited in Kyoto University, was used for transformation. The GY medium (pH 6.5) containing 2% glucose and 1% Bacto yeast extract (Becton, Dickinson and Company, Sparks, MD, USA) was used for routine cultivation and as a selective medium by the addition of antibiotics. To collect M. alpina 1S-4 spores, the Czapek-Dox medium was used as described previously (16). All fungal cultivations were performed at 28°C. Escherichia coli DH5α was used for DNA manipulation and cultivated at 37°C under vigorous shaking (300 rpm). Construction of transformation vector, pDZeo and pDZeoGLELO Vector pDura5 (12) was previously constructed by the modification of vector pD4, which had been used for the transformation of M. alpina CBS 224.37, and which was kindly supplied by Dr. Archer, D.B. (University of Nottingham, UK). The PCR amplification of a modified Zeocin-resistance gene was carried out in a total volume of 30 µl containing 200 ng of pEM7/Zeo (Invitrogen), 0.2 µl of LA Taq (Takara Bio), 10 ×LA Taq buffer, 2 mM MgCl2, 10 pmol of primers and 200 µM of each dNTP. Two primers, ZeoFNcoI (5′-AAACCATGGCCAAGTTGACCAGT-3′) and ZeoRxEoB (5′-AGAGGATCCCCGGGAATTATCAGT-3′), were designed on the basis of the Zeocin resistance gene of pEM7/Zeo and had annealing temperatures of being 61.6°C. These two primers contained a NcoI restriction site and a BamHI restriction site, respectively (underlined). The resulting 400-bp fragment was cloned into the pT7 Blue T-vector (Novagen, Madison, WI, USA), the resulting vector was designated as pT7-Zeo, and the sequence was confirmed. The modified Zeocin-resistance gene cut out from pT7-Zeo with NcoI and BamHI was ligated to pDura5 (without the ura5 gene) digested with NcoI and BamHI to construct pDZeo for the transformation of M. alpina by microprojectile bombardment (Fig. 1). The transformation vector pDZeoGLELO was constructed so that the expression system of a homologous GLELO gene (AB193123) was integrated into pDZeo. The expression unit was constructed as described previously (8). The expression unit was digested with EcoRI and ligated to pDZeo digested with the same enzyme to generate pDZeoGLELO (Fig. 1). Transformation of M. alpina with pDZeo and pDZeoGLELO by microprojectile bombardment A PDS-1000/He particle delivery system (Bio-Rad Laboratories, Hercules, CA, USA) was used for the transformation under the same conditions as described previously (12). In the case of transformation with pDZeo, 4.0 ×106 spores were spread on a plate containing the GY medium without Zeocin. After the bombardment, the plate was statically incubated at 12°C or 28°C for 0–15 h. Swollen spores, just prior to germination, were collected by suspending them in ~500 µl of Tween 80 solution (0.3 µl/ml of distilled H2O), and then spread on the selective GY medium containing 20 mg/ml Zeocin, followed by incubation at
FIG. 1. Maps of M. alpina 1S-4 transformation vector pDZeo and pDZeoGLELO. The details of the genes and the method used to construct the vector are given in Materials and Methods. Zeo, Zeocin resistance gene; bla, ampicillin resistance gene; his H4.1p, M. alpina histone H4.1 promoter fragment from strain CBS 528.72; trpCt, Aspergillus nidulans N-(5′-phosphoribosyl)anthranilate isomerase (trpC) transcription terminator; rDNA, M. alpina 1S-4 18S rDNA fragment. Restriction sites: E, EcoRI; N, NcoI; B, BamHI; X, XbaI; H, HindIII; S, SspI.
28°C. After 2 d, transformants resistant to Zeocin were isolated and maintained in the same medium as that for cultivation under selective conditions for further analyses. To select stable transformants, the following method was adopted: Individual mycelia were inoculated onto the GY medium without Zeocin. After 2 d, a mycelium was picked up from the outer part of a colony and then inoculated onto a fresh GY medium. The selection was repeated three times. Mycelia from the third culture were finally inoculated onto the GY medium containing 20 mg/ml Zeocin. Transformants grown on the selection medium were defined as being stable with Zeocin-resistance. In the case of transformation with pDZeoGLELO, intact spores of M. alpina 1S-4 wild-type strain were harvested from the surface of the Czapek–Dox agar medium (2.0×108 spores/300 cm2). Spores (4.0 ×107) were spread on a GY medium plate without Zeocin. Each plate was bombarded twice with a PDS-1000/He particle
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delivery system. After the bombardment, the plate was statically incubated at 12°C for 8–25.5 h. Swollen spores were collected by suspending them in ~500 ml of distilled H2O, spread onto the selective GY medium containing 20 mg/ml Zeocin, and then incubated at 28°C. After 2 d, mycelia were isolated and maintained in the same medium under selective conditions for further analyses. Stable transformants were selected by the same methods as in the case of pDZeo transformants. Confirmation of transformation by PCR Genomic DNA of transformants was obtained from mycelia grown in the GY medium. Genomic DNA of M. alpina strains was prepared according to a method described previously (12). Transformants were confirmed by PCR using the forward primer ZeoFNcoI and the reverse primer ZeoRxEoB. The integration of plasmid pDZeo or pDZeoGLELO into an appropriate chromosomal rDNA region was verified using the vector-specific forward primer RDNA1 (5′-ACAGGTACACTTGTTTAGAG-3′) and the reverse primer RDNA2 (5′-CGCTGCGTTCTTCATCGATG-3′), both of which were designed with reference to the report of Mackenzie et al. (17). RDNA1 and RDNA2 respectively anneal immediately upstream of the XbaI site in the trpC terminator region and the 5.8S rDNA gene; this gene lies downstream of the 18S rDNA region in the genomic DNA but not in these vectors. In the case of pDZeoGLELO transformants, the forward primer GLELOF (5′-CACCATGGAGTCGATTGCGC-3′) and the reverse primer GLELOR (5′-GTGGATCCTTACTGCAACTTCCTTGCCTT-3′) were also available to detect GLELO cDNA in the genomic DNA (8). Fatty acid analyses The spores of M. alpina recombinant cells transformed with pDZeoGLELO and those of wild-type cells (host cells) were inoculated into a 20-ml Erlenmeyer flask containing 5 ml of GY medium consisting of 2% glucose and 1% yeast extract. The culturing was carried out at 28°C with reciprocal shaking (120 strokes/min) for 10 d. Fatty acid analysis was performed basically as described in a previous paper (9).
RESULTS Screening of antibiotics that inhibit growth of M. alpina 1S-4 The first step was to screen for useful antibiotics that inhibit the growth of M. alpina 1S-4. Growth experiments were carried out on the GY agar medium containing various concentrations of antibiotics. As a result, Zeocin was considered to be a possible candidate (data not shown): other antibiotics, such as blasticidin S, bleomycin, Geneticin, hygromycin B, and oligomycin did not inhibit the growth of the strain, whereas a high concentration of Zeocin (20 mg/ml) did, although not completely. However, the germination of spores was completely inhibited by Zeocin. Since the transformation system used in this study involved gene transfer into spores, Zeocin was considered suitable. The concentration of Zeocin was fixed at 20 mg/ml unless otherwise stated. Transformation of M. alpina 1S-4 and optimization of conditions The M. alpina 1S-4 transformation vector pDZeo was developed by means of the methods as described in Materials and Methods. pDZeo was designed to be integrated into the rDNA locus of M. alpina 1S-4 genomic DNA. When pDZeo was bombarded into the spores on the GY medium containing Zeocin (selective medium), mycelia did not appear, suggesting that the integration of pDZeo into the genomic DNA and the expression of the Zeocin-resistance gene must have occurred in the host cells
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TABLE 1. Effects of preincubation temperature and period on transformation frequency prior to transfer to selective medium Preincubation Number of transforTemp. Period Morphological appearance of spores mantsa (°C) (h) 12 0 Small and rod-shaped, before germination –b 6 Small and rod-shaped, before germination 9 10 Swollen and spherical, before germination 14 15 ~50% of spores germinated 10 21 All spores germinated N.D.c 28 0 Small and rod-shaped, before germination – 6 Swollen and spherical, before germination 12 10–21 All spores germinated N.D. Bombarded spores were grown on the GY medium without Zeocin at 12°C or 28°C for the designated period (preincubation). After the cultivation, spores were collected and transferred to the Zeocin-containing selective medium. Morphological appearances of spores just prior to the transfer were observed under a microscope. a The number of transformants was determined by counting colonies on the Zeocin-containing selective medium. b –, Not detected. c N.D., Not determined.
before they were placed on the Zeocin-containing medium. Therefore, once gene transfer into the spores had occurred on the GY medium, and cultivation at 12°C for 2 d to allow germination and some growth had been performed, the GY medium containing Zeocin was poured onto each plate. As a result, the surfaces of the plates were covered with numerous mycelia that could not be selected. On the basis of the finding that spores never grew on Zeocin-containing medium and the mycelia were hardly affected by Zeocin, the following method was examined: Vector-bombarded spores on the GY medium were preincubated just prior to germination, and then the spores were collected from the surface of each plate, and spread onto the selective medium, and then cultivated at 28°C. As a result, many transformants were isolated. We considered that preincubation must be the most important step, and thus optimized preincubation temperature and period (Table 1). The highest transformation frequency was obtained when the preincubation was at 12°C for 10 h, and spores were fully swollen just prior to germination in this state. At 12°C for 21 h or at 28°C for 10–21 h, all the spores completely germinated and stuck to the surface of the medium. Up to seven strains were randomly isolated from each selective medium, and the strains were characterized by growth and genetic analyses. Analyses of stability of transformants and comfirmation of transformation by PCR Stable transformants were selected as described in Materials and Methods. Out of 26 transformants obtained, regardless of the preincubation conditions, 15 were judged to be stable. The appearance rate of stable transformants reached about 60% regardless of the conditions. Secondly, of the 15 stable transformants, four (#1, 6, 13, and 28) were selected for further analyses, because all four could grow vigorously under the Zeocincontaining conditions (up to 20 mg/ml) (Fig. 2). Genomic DNA was prepared from each transformant cultivated in the GY liquid medium. The strategy for transformation with pDZeo was based on homologous integration into rDNA. Transformed cells, therefore, were confirmed by PCR using the primers RDNA1 and RDNA2. A band corre-
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FIG. 2. Growth of wild-type strain and transformants on GY medium with or without Zeocin. Each mycelium was inoculated onto the GY medium with or without Zeocin, and then incubated at 28°C for 4 d.
sponding to a 1.5-kb amplified fragment appeared on an agarose gel only when pDZeo was integrated into the chromosomal rDNA locus. For further confirmation, the vectorspecific reverse primer ZeoRxEoB was also used with the forward primer ZeoFNcoI to confirm the presence of pDZeo in transformants. As shown in Fig. 3, the 1.5-kb amplified fragment obtained with RDNA1 and RDNA2 indicated that pDZeo existed at the rDNA locus of the genomic DNAs of the transformants. On the other hand, a nonspecific band corresponding to a 2.8-kb fragment was observed for all transformants and host cells. This nonspecific band was determined to represent a certain rDNA region amplified with these two primers: primer RDNA1 anneals at this unknown region of 18S rDNA, leading to the generation of this nonspecific band (data not shown). In the cases of #6 and #28, the intensity of the nonspecific bands was stronger than that of bands corresponding to 1.5-kb fragments. This tendency has frequently been observed in unstable transformants (data not shown). Transformation of M. alpina 1S-4 with pDZeoGLELO We reported that GLELO gene overexpression leads to a high productivity of AA in the ura5– strain (8). In the present study, we tried to overexpress the gene in wild-type 1S-4. Wild-type M. alpina with pDZeoGLELO was transformed as described in Materials and Methods. Out of 60 transformants obtained, two stable transformants (#55 and #63) were selected. The transformation frequency was 6.8 transformants/µg of transformation vector. Both transformants could grow on the GY medium containing 20 mg/ml Zeocin more vigorously than the wild-type strain (data not shown). The transformation was confirmed by PCR (data
FIG. 3. Confirmation of transformation by PCR. Transformation was confirmed by PCR using two primers, ZeoFNcoI/ZeoRxEoB (top) and RDNA1/RDNA2 (bottom). M denotes 2-log DNA ladder markers (New England BioLabs).
not shown). In addition, real-time quantitative PCR analysis showed that the quantity of GLELO RNA in transformant #63 was 3.4-fold higher than that in the host strain (wildtype strain) on the 4th day (data not shown). To compare fatty acid productivities, these two transformants were grown in the GY medium at 28°C for 10 d and fatty acid analyses were carried out (Table 2). Marked differences in growth were not observed. However, the content of AA in the pDZeoGLELO transformants was higher than that in the host strain and pDZeo transformants. GLA content in the pDZeoGLELO transformants decreased concomitantly with the increase in AA content. DISCUSSION In our previous study (12), we established a basic transformation system for M. alpina using its uracil auxotrophic mutants. The system could only be applied to uracil auxotrophs. We then attempted to construct a universal system
TABLE 2. Comparison of PUFA productivities of M. alpina 1S-4, pDZeo transformant, and two pDZeoGLELO transformants Growtha Total FAb (mg/ml) (mg/ml)
FA composition (wt%) 16:0 18:0 18:1n-9 18:2n-6 GLA DGLA AA Others 9.18±0.02 4.70±0.47 10.4±0.21 3.34±0.24 14.3±0.10 6.73±0.27 5.09±0.07 3.94±0.09 53.4±0.98 2.88±0.01 Wild-type 1S-4 pDZeo transformant #1 9.66±0.08 6.18±0.10 10.4±0.28 3.99±0.15 10.1±0.25 6.61± 0.11 4.32±0.00 3.99±0.09 58.2±0.86 2.39±0.04 pDZeoGLELO transformant #55 9.91±0.11 6.88±0.30 9.96±0.33 3.99±0.53 8.24±0.48 5.5±0.06 4.02±0.22 3.93±0.11 62.2±1.12 2.18±0.07 pDZeoGLELO transformant #63 9.72±0.02 7.09±0.95 8.29±0.21 2.37±0.28 6.12±0.32 4.12±0.04 3.41±0.05 3.23±0.40 71.2±1.34 1.31±0.27 All strains were grown at 28°C for 10 d in 5 ml of the GY medium under reciprocal shaking (120 strokes/min). These values are the means of duplicate experiments. a Milligram of dry cell weight/ml of culture broth. b Milligram of total fatty acid/ml of culture broth. FA, Fatty acid; 16:0, palmitic acid; 18:0, stearic acid; 18:1n-9, oleic acid; 18:2n-6, linoleic acid; GLA, γ-linolenic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid. Strains
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for the wild-type strain, M. alpina 1S-4, and various mutants, which had been isolated, characterized and utilized for PUFA production, and for the study of PUFA metabolism. Antibiotics generally show a strong inhibitory effect on prokaryote growth, but not always on eukaryotes such as fungi. Mackenzie et al. reported transformation systems using M. alpina CBS 224.37, which is sensitive to hygromycin B (17). Although the morphological shape of M. alpina 1S-4 is very similar to that of M. alpina CBS 224.37, M. alpina 1S-4 is not sensitive to hygromycin B even at high concentrations. Out of some antibiotics reported to be effective against filamentous fungi, we found that Zeocin completely inhibited the germination of spores of M. alpina 1S-4. In this study, we developed another transformation method using Zeocin as a selectable marker, which might be useful in the development of transformation systems for other organisms. We consider that the selection of stable transformants is the most important step for further analysis and practical studies. From the application viewpoint, PUFA production is performed in submerged cultures and the GY medium has been reported to be the most suitable medium for this (18, 19). The cultivation of unstable transformants in a Zeocincontaining medium to generate selective pressure leads to high costs and biohazard problems, but stable transformants require no continuous selective pressure during cultivation. In the previous transformation method using uracil auxotrophs on a uracil-free synthetic medium, the appearance rate of stable transformants was about 10% for all isolates. On the other hand, we obtained them at a rate of 60% in this study. The lower rate for uracil auxotroph transformation may be attributed to the random mutation occurring by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment. Consequently, we obtained stable transformants resistant to Zeocin at a higher rate. We analyzed differences in genetic property and growth among four stable transformants (#1, 6, 13, and 28). They could grow on the GY medium containing 20 mg/ml Zeocin after serial cultivation on the GY medium. Their growth rate was determined to be the same as that of the wild-type strain in the absence of Zeocin (Fig. 2). This observation is suitable for practical applications, as mentioned above. In addition, genetic analyses revealed some differences among the transformants (Fig. 3). Two PCR products, 1.5-kb and 2.8-kb fragments, were detected for all samples when the amplification was performed using RDNA1 and RDNA2. The 2.8-kb fragment was identified as a certain region in the rDNA locus (data not shown). The ratio of the amounts of the fragments differed among individual transformants. This difference cannot be explained but the ratio affected the degree of stability, that is, transformants with higher amounts of the 1.5-kb fragment were defined as stable. Therefore, #1 and #13 were defined as more stable transformants than #6 and #28. The copy number of the transformation vector in the transformants was not determined, but it is clear that #1 and #13 contain more GLELO genes in their genomic DNA than #6 and #28 as shown in Fig. 3. We investigated the fatty acid compositions of pDZeo and pDZeoGLELO transformants (Table 2). pDZeo trans-
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formant #1, as the control strain, showed the same rate of growth as the wild-type 1S-4. Therefore, it is considered that the expression of the marker gene does not affect growth. On the other hand, the total fatty acid content of the pDZeo transformant was higher than that of the wild-type 1S-4. The reason remains unclear, but one hypothesis can be proposed. Wynn et al. reported that the exhaustion of the nitrogen source triggers lipid accumulation (20). Overproduction of a Zeocin-resistant protein may accelerate the exhaustion of the nitrogen source, leading to a higher lipid accumulation. However, the control strain showed a fatty acid composition comparable to that of the host cells. The results provide important information for further study. The fatty acid compositions of two GLELO-overexpressing transformants were different compared with those of the host cells and pDZeo transformants. In the case of pDZeoGLELO transformant #63, the AA composition was 1.2-fold higher than that of the pDZeo transformants. In addition, the compositions of other fatty acids generally decreased, which suggests a prompt fatty acid modification to AA. We previously obtained an equal increase in the rate of AA production using ura5– strain as the host (8). However, the productivity of pDZeoGLELO transformant #63 has never been obtained. The present technique led to an increase in AA productivity of the wild-type strain (from 2.51 to 5.05 mg of AA/ml of culture broth). The increased productivity is thought to be derived from the overexpression of the GLELO gene. The previous system using ura5– strains as the host (8) did not realize such a high productivity of AA; thus the present system is useful for increasing the fatty acid production. In this study, we succeeded in establishing of an overall transformation system for this fungus, and in improving the fatty acid composition. It is expected that genetic analyses and approaches will clarify the biosynthetic pathway of AA and contribute to the development of the oil industry. We aim to overexpress or destroy a gene involved in PUFA biosynthesis for the control of PUFA metabolism. ACKNOWLEDGMENTS We wish to thank Dr. David B. Archer (School of Life and Environmental Sciences, University of Nottingham, United Kingdom) for providing the Mortierella transformation vector. This work was supported in part by the New Energy and Industrial Technology Development Organization (NEDO), and a Grant-in-Aid for Scientific Research (no. 15658024 for S.S.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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