Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e3, 2014 www.elsevier.com/locate/jbiosc
TECHNICAL NOTE
Construction of transformation system in Penicillium purpurogenum Ryo Kojima, Teppei Arai, Takafumi Kasumi, and Jun Ogihara* Department of Chemistry and Life Science, Nihon University College of Bioresource Sciences, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan Received 3 April 2014; accepted 31 August 2014 Available online xxx
Penicillium purpurogenum attracts attention in the food industry and biomass degradation. We expressed green fluorescent protein (GFP) with pBPE, a novel vector, and constructed a transformation system for P. purpurogenum. The accumulation of GFP was confirmed by fluorescence microscopy. In future, this system may prove useful for the genetic modification of P. purpurogenum. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Penicillium purpurogenum; Transformation; Green fluorescent protein; Protoplasts; Promoter; Terminator]
In East Asia, Monascus pigments are used as a natural food colorant. Recent studies showed that pigments produced by Monascus spp. possess antibacterial, anticancer and antioxidation activities (1e3). However, some strains of Monascus produce citrinin, a nephrotoxic mycotoxin, as a byproduct of pigment biosynthesis (4). For this reason, the use of Monascus pigments is banned in the European Union (5). Penicillium purpurogenum IAM15392 produces PPO, PP-Y, PP-V and PP-R in the medium under specific conditions (6e8). The structure of these compounds is similar to that of Monascus pigment (6,8). Moreover, to date there have been no reports of citrinin production by this fungus, suggesting that P. purpurogenum could be a potentially valuable commercial source of natural food colorant (9). In addition, this fungus is used to produce xylanase and b-glucosidase (10,11), which catalyze the hydrolysis of xylan and cellulose, respectively. P. purpurogenum is therefore useful not only in the food industry, but also for biomass degradation. Genetic modification can be used to create useful mutants of P. purpurogenum (e.g., strains that produce large amounts of useful compounds, transformants useful for elucidating biosynthesis pathways, and mutants that can regulate the biosynthesis of toxic compounds). All these approaches require the induction of gene expression, overexpression, RNAi and homologous recombination. However, to date, no method has been demonstrated for the transformation of P. purpurogenum, although it has reported in Penicillium spp. such as Penicillium chrysogenum, P. expansum, and P. nordicum (12e14). We therefore constructed a green fluorescent protein (GFP) expression vector, pBPE-GFP, and aimed to transform P. purpurogenum IAM15392 by using it. pBlue script II SKþ was used to construct pBPE (Fig. 1). Here, to exert higher selective pressure, two drug-resistant genes were introduced in this vector. A hygromycin-resistant gene cassette and a geneticin-resistant gene cassette were inserted into the ApaI and SacI site of pBlue Script II SKþ, respectively. The trpC promoter and
* Corresponding author. Tel./fax: þ81 466 84 3945. E-mail address: [email protected] (J. Ogihara).
terminator were amplified by PCR with specific primers containing the XhoI and ApaI sites, respectively. The PCR promoter and terminator products were inserted into the NotI and XhoI sites of pBlue script II SKþ, respectively. The multi-cloning site was inserted into the XhoI/ApaI site of this vector. pBPE-GFP was constructed by introducing GFP into the EcoRV site of pBPE, then GFP was amplified using specific primers(GFP-sense 50 -TATATCATGGCCGACAAGCA-30 , GFP-antisense 50 -GAACTCCAGCAGGACCATGT-30 ). PCR was performed using the general reaction conditions recommended for Go taq (Promega Corporation, Madison, WI, USA). The 7.2 106 spores of P. purpurogenum IAM15392 was grown in four, 500-mL Erlenmeyer flasks containing 100 mL of PP-O production medium (20 g of soluble starch, 2 g of yeast extract per L of 50 mM citric acid/Na3 citrate buffer, pH 5.0) for 24 h at 30 C with shaking at 130 rpm. The mycelia were harvested by centrifuging for 15 min at 4 C, 1600 g. The pellet washed with 25 mL of 0.8 M NaCl for 15 min at 4 C, then centrifuged (1600 g). Washing/centrifugation was repeated twice, then the pellet was transferred to a 15 mL tube. Subsequently, 5 mL of YatalaseÔ enzyme solution (final concentration 1.5%; Takara, Otsu, Japan in 0.8 M NaCl, 1 mM DTT, 0.01 M Na phosphate buffer, pH 6.0) was added and the sample was incubated at 30 C for 90 min. The resulting protoplasts were filtered through a sterile miracloth and transferred to a 50 mL tube. The filtrate was centrifuged for 10 min at 4 C, 1600 g, and the supernatant was removed. The protoplasts were washed with 10 mL of solution I (final concentration of 0.8 M NaCl, 0.05 M CaCl2, and 0.01 M Tris/HCl, pH 7.5), resuspended, and centrifuged for 15 min at 4 C, 1600 g; the wash/centrifugation process was then repeated. The 300 mL of solution I, 0.45 mL of 1 M DTT, and 150 mL of solution II (final concentration of 50% polyethylene glycol 4000, 0.05 M CaCl2 and 0.01 M Tris/HCl, pH 7.5) were added. pBPE-GFP (5 mg or 10 mg) were transfected into 100 mL of protoplasts, then the sample was incubated for 30 min on ice. Solution II (500 mL) was added, the protoplasts were suspended, then incubated for 20 min on ice. Solution I (10 mL) was added and the tube was inverted several times. After centrifuging for 5 min at 4 C, 1600 g, the supernatant was decanted, then it was left slightly to suspend the
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.08.016
Please cite this article in press as: Kojima, R., et al., Construction of transformation system in Penicillium purpurogenum, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.08.016
2
KOJIMA ET AL.
FIG. 1. The plasmid map of pBPE and original restriction site. pBPE contains two drug resistant genes. pBPE-GFP was constructed by introducing GFP fragment amplified by PCR into EcoRV site of multi cloning site of pBPE. trpC promoter and terminator were used for expression of geneticin resistant gene, hygromycin resistant gene, and target gene in multi cloning site, respectively. Geneticinr, geneticin resistant gene; PtrpC, trpC promoter; MCS, multi cloning site; TtrpC, trpC terminator; Hygr, hygromycin resistant gene; Ampr, ampicillin resistant gene.
J. BIOSCI. BIOENG., pellet. Following the manipulation, 50 mL of protoplast suspension was plated onto YMA (5 g of peptone, 3 g of yeast extract, 3 g of malt extract, 10 g of glucose, and 20 g of agar per L) containing a final concentration of 3.8% NaCl as an osmotic stabilizer and 150 mg/mL G418 disulfate aqueous solution (Nacalai Tesque, Kyoto, Japan). YM containing a final concentration of 0.5% agar (gelling temperature 30e31 C; Nacalai Tesque), 3.8% NaCl, and 150 mg/mL G418 was overlaid on protoplasts. The plates were incubated at 30 C until colonies were visible, then growth of geneticin resistant colonies was examined on YMA plate containing 150 mg/mL G418 or without its drug. Wild type and transformants grown on YMA plate at 30 C, for 7 days. GFP fluorescence was imaged using a Typhoon 9410 imager (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire , UK) using a 488 nm laser for excitation and a 526 nm short pass (SP) filter to collect the emission. GFP expression was observed using a fluorescence microscope (Keyence, Osaka, Japan). The pBPE sequence inserted in the genome was detected by PCR using GFPsense and GFP-antisense as vector-specific primers. Growth of selected colonies were evaluated (Fig. S1). Wild type and all drug resistant strains grew better in YMA. Moreover, drug resistant strains also could grow in its medium containing G418 although growth of wild type was inhibited. From these results, it was found that six strains are able to grow stably in presence of G418. GFP expression was detected (Fig. 2A). TP6, TP7, and TP8 exhibited high signal intensity, whereas no signal was detected
FIG. 2. (A) GFP expression in the transformants. Typhoon 9410 was used for detection of GFP signal. The wild type and transformants were grown on YMA at 30 C, for 7 days. W indicates wild type and TP indicates transformants of P. purpurogenum IAM15392. (B) Localization of GFP. The wild type and TP6 were grown on PD at 30 C, for 4 days. GFP expression was confirmed by fluorescence microscopy. (a, c) Bright field images of the transformants. (b, d) GFP expression in panels a and c, respectively. (e, f) Images of wild type. (a, b, e, f) Microscope magnification 400. (c, d) Microscope magnification 200. Bars: 50 mm.
Please cite this article in press as: Kojima, R., et al., Construction of transformation system in Penicillium purpurogenum, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.08.016
VOL. xx, 2014
TECHNICAL NOTE
TABLE 1. The result of transformation in P. purpurogenum IAM15392. Amount of pBPE-GFP (mg) 5 10
Total colonies
Number of transformants
Percentage of transformants (%)a
21 24
0 3
0 12.5
3
industry and for biomass degradation. Hence, work should continue to develop an efficient transformation system and vector providing higher gene expression. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2014.08.016.
a Percentage of transformants was regarded as number of GFP fluorescence strains/total colonies.
References from TP1, TP2, and TP13. Three transformants were thus obtained. The location of GFP in T6 was observed using fluorescence microscopy (Fig. 2B) and determined to be accumulated in the hypha. Ideally, GFP mutants should be grown in medium in which no pigment is produced because pigments produced by P. purpurogenum IAM15392 cause intrinsic fluorescence. These results showed that the trpC promoter and terminator from Aspergillus nidulans can be used in P. purpurogenum. Both geneticin and hygromycin worked as selectable markers, but we found that geneticin has higher selective pressure than hygromycin (data not shown). Furthermore, although many false positive colonies were obtained using PDA and PP-O production medium containing hygromycin B (Wako, Osaka, Japan) (data not shown), few false positives (data not shown) were obtained using YMA containing hygromycin B, suggesting that the selective pressure of this drug depends on the medium. YMA medium was better for obtaining transformants of P. purpurogenum IAM15392. Transformation data are summarized in Table 1. Either 5 mg or 10 mg of pBPE-GFP were used to transform the fungus. In both cases the total number of colonies were almost the same, but the ratio of transformants to it was vastly different. When 5 mg of pBPE-GFP was used, the rate was 0%, whereas the rate was 12.5% when 10 mg of the vector was used. Transformation requires favorable conditions. For example, the number of stable colonies depends on quantity of plasmid in A. nidulans (15). Moreover, it has been reported that the highest co-transformation efficiency (7%) was observed in Penicillium griseoroseum when the ratio of two plasmids was 3:6; when equal amounts of the two plasmids were used, co-transformation efficiency decreased (16). These results suggest that 10 mg provides more transformants than 5 mg in P. purpurogenum. However, only three transformants among 24 geneticin resistant colonies obtained in 10 mg. It is possible that this is because geneticin resistance in the host was newly induced by spontaneous mutation or that geneticin resistant strains with no fluorescence have not been introduced with GFP gene but with the drug resistant gene only although a strain having both GFP genes and geneticin resistant gene is preferred. In summary, although we could construct a transformation system for P. purpurogenum, improvements to this method are required to increase transformation efficiency. In future, genetic modification of P. purpurogenum may increase its utility in the food
1. Kim, C., Jung, H., Kim, Y. O., and Shin, C. S.: Antimicrobial activities of amino acid derivatives of monascus pigments, FEMS Microbiol. Lett., 264, 117e124 (2006). 2. Zheng, Y., Xin, Y., Shi, X., and Guo, Y.: Anti-cancer effect of rubropunctatin against human gastric carcinoma cells BGC-823, Appl. Microbiol. Biotechnol., 88, 1169e1177 (2010). 3. Akihisa, T., Tokuda, H., Yasukawa, K., Ukiya, M., Kiyota, A., Sakamoto, N., Suzuki, T., Tanabe, N., and Nishino, H.: Azaphilones, furanoisophthalides, and amino acids from the extracts of Monascus pilosus-fermented rice (red-mold rice) and their chemopreventive effects, J. Agric. Food Chem., 53, 562e565 (2005). 4. Wang, Y. Z., Ju, X. L., and Zhou, Y. G.: The variability of citrinin production in Monascus type cultures, Food Microbiol., 22, 145e148 (2005). 5. Mapari, S. A., Thrane, U., and Meyer, A. S.: Fungal polyketide azaphilone pigments as future natural food colorants? Trends Biotechnol., 28, 300e307 (2010). 6. Ogihara, J. and Oishi, K.: Effect of ammonium nitrate on the production of PPV and monascorubrin homologues by Penicillium sp. AZ, J. Biosci. Bioeng., 93, 54e59 (2002). 7. Ogihara, J., Kato, J., Oishi, K., Fujimoto, Y., and Eguchi, T.: Production and structural analysis of PP-V, a homologue of monascorubramine, produced by a new isolate of Penicillium sp. J. Biosci. Bioeng., 90, 549e554 (2000). 8. Ogihara, J., Kato, J., Oishi, K., and Fujimoto, Y.: PP-R, 7-(2-hydroxyethyl)monascorubramine, a red pigment produced in the mycelia of Penicillium sp. AZ, J. Biosci. Bioeng., 91, 44e47 (2001). 9. Dufossé, L., Fouillaud, M., Caro, Y., Mapari, S. A., and Sutthiwong, N.: Filamentous fungi are large-scale producers of pigments and colorants for the food industry, Curr. Opin. Biotechnol., 26, 56e61 (2014). 10. Belancic, A., Scarpa, J., Peirano, A., Díaz, R., Steiner, J., and Eyzaguirre, J.: Penicillium purpurogenum produces several xylanases: purification and properties of two of the enzymes, J. Biotechnol., 41, 71e79 (1995). 11. Hidalgo, M., Steiner, J., and Eyzaguirre, J.: Beta-glucosidase from Penicillium purpurogenum: purification and properties, Biotechnol. Appl. Biochem., 15, 185e191 (1992). 12. Stahl, U., Leitner, E., and Esser, K.: Transformation of Penicillium chrysogenum by a vector containing a mitochondrial origin of replication, Appl. Microbiol. Biotechnol., 26, 237e241 (1987). 13. Buron-Moles, G., López-Pérez, M., González-Candelas, L., Viñas, I., Teixidó, N., Usall, J., and Torres, R.: Use of GFP-tagged strains of Penicillium digitatum and Penicillium expansum to study host-pathogen interactions in oranges and apples, Int. J. Food Microbiol., 160, 162e170 (2012). 14. Schmidt-Heydt, M., Schunck, T., and Geisen, R.: Expression of a gfp gene in Penicillium nordicum under control of the promoter of the ochratoxin A polyketide synthase gene, Int. J. Food Microbiol., 133, 161e166 (2009). 15. Yelton, M. M., Hamer, J. E., and Timberlake, W. E.: Transformation of Aspergillus nidulans by using a trpC plasmid, Proc. Natl. Acad. Sci. USA, 81, 1470e1474 (1984). 16. Lopes, F. J., de Araújo, E. F., and de Queiroz, M. V.: Easy detection of green fluorescent protein multicopy transformants in Penicillium griseoroseum, Genet. Mol. Res., 3, 449e455 (2004).
Please cite this article in press as: Kojima, R., et al., Construction of transformation system in Penicillium purpurogenum, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.08.016
TECHNICAL NOTE
Construction of transformation system in Penicillium purpurogenum Ryo Kojima, Teppei Arai, Takafumi Kasumi, and Jun Ogihara* Department of Chemistry and Life Science, Nihon University College of Bioresource Sciences, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan Received 3 April 2014; accepted 31 August 2014 Available online xxx
Penicillium purpurogenum attracts attention in the food industry and biomass degradation. We expressed green fluorescent protein (GFP) with pBPE, a novel vector, and constructed a transformation system for P. purpurogenum. The accumulation of GFP was confirmed by fluorescence microscopy. In future, this system may prove useful for the genetic modification of P. purpurogenum. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Penicillium purpurogenum; Transformation; Green fluorescent protein; Protoplasts; Promoter; Terminator]
In East Asia, Monascus pigments are used as a natural food colorant. Recent studies showed that pigments produced by Monascus spp. possess antibacterial, anticancer and antioxidation activities (1e3). However, some strains of Monascus produce citrinin, a nephrotoxic mycotoxin, as a byproduct of pigment biosynthesis (4). For this reason, the use of Monascus pigments is banned in the European Union (5). Penicillium purpurogenum IAM15392 produces PPO, PP-Y, PP-V and PP-R in the medium under specific conditions (6e8). The structure of these compounds is similar to that of Monascus pigment (6,8). Moreover, to date there have been no reports of citrinin production by this fungus, suggesting that P. purpurogenum could be a potentially valuable commercial source of natural food colorant (9). In addition, this fungus is used to produce xylanase and b-glucosidase (10,11), which catalyze the hydrolysis of xylan and cellulose, respectively. P. purpurogenum is therefore useful not only in the food industry, but also for biomass degradation. Genetic modification can be used to create useful mutants of P. purpurogenum (e.g., strains that produce large amounts of useful compounds, transformants useful for elucidating biosynthesis pathways, and mutants that can regulate the biosynthesis of toxic compounds). All these approaches require the induction of gene expression, overexpression, RNAi and homologous recombination. However, to date, no method has been demonstrated for the transformation of P. purpurogenum, although it has reported in Penicillium spp. such as Penicillium chrysogenum, P. expansum, and P. nordicum (12e14). We therefore constructed a green fluorescent protein (GFP) expression vector, pBPE-GFP, and aimed to transform P. purpurogenum IAM15392 by using it. pBlue script II SKþ was used to construct pBPE (Fig. 1). Here, to exert higher selective pressure, two drug-resistant genes were introduced in this vector. A hygromycin-resistant gene cassette and a geneticin-resistant gene cassette were inserted into the ApaI and SacI site of pBlue Script II SKþ, respectively. The trpC promoter and
* Corresponding author. Tel./fax: þ81 466 84 3945. E-mail address: [email protected] (J. Ogihara).
terminator were amplified by PCR with specific primers containing the XhoI and ApaI sites, respectively. The PCR promoter and terminator products were inserted into the NotI and XhoI sites of pBlue script II SKþ, respectively. The multi-cloning site was inserted into the XhoI/ApaI site of this vector. pBPE-GFP was constructed by introducing GFP into the EcoRV site of pBPE, then GFP was amplified using specific primers(GFP-sense 50 -TATATCATGGCCGACAAGCA-30 , GFP-antisense 50 -GAACTCCAGCAGGACCATGT-30 ). PCR was performed using the general reaction conditions recommended for Go taq (Promega Corporation, Madison, WI, USA). The 7.2 106 spores of P. purpurogenum IAM15392 was grown in four, 500-mL Erlenmeyer flasks containing 100 mL of PP-O production medium (20 g of soluble starch, 2 g of yeast extract per L of 50 mM citric acid/Na3 citrate buffer, pH 5.0) for 24 h at 30 C with shaking at 130 rpm. The mycelia were harvested by centrifuging for 15 min at 4 C, 1600 g. The pellet washed with 25 mL of 0.8 M NaCl for 15 min at 4 C, then centrifuged (1600 g). Washing/centrifugation was repeated twice, then the pellet was transferred to a 15 mL tube. Subsequently, 5 mL of YatalaseÔ enzyme solution (final concentration 1.5%; Takara, Otsu, Japan in 0.8 M NaCl, 1 mM DTT, 0.01 M Na phosphate buffer, pH 6.0) was added and the sample was incubated at 30 C for 90 min. The resulting protoplasts were filtered through a sterile miracloth and transferred to a 50 mL tube. The filtrate was centrifuged for 10 min at 4 C, 1600 g, and the supernatant was removed. The protoplasts were washed with 10 mL of solution I (final concentration of 0.8 M NaCl, 0.05 M CaCl2, and 0.01 M Tris/HCl, pH 7.5), resuspended, and centrifuged for 15 min at 4 C, 1600 g; the wash/centrifugation process was then repeated. The 300 mL of solution I, 0.45 mL of 1 M DTT, and 150 mL of solution II (final concentration of 50% polyethylene glycol 4000, 0.05 M CaCl2 and 0.01 M Tris/HCl, pH 7.5) were added. pBPE-GFP (5 mg or 10 mg) were transfected into 100 mL of protoplasts, then the sample was incubated for 30 min on ice. Solution II (500 mL) was added, the protoplasts were suspended, then incubated for 20 min on ice. Solution I (10 mL) was added and the tube was inverted several times. After centrifuging for 5 min at 4 C, 1600 g, the supernatant was decanted, then it was left slightly to suspend the
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.08.016
Please cite this article in press as: Kojima, R., et al., Construction of transformation system in Penicillium purpurogenum, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.08.016
2
KOJIMA ET AL.
FIG. 1. The plasmid map of pBPE and original restriction site. pBPE contains two drug resistant genes. pBPE-GFP was constructed by introducing GFP fragment amplified by PCR into EcoRV site of multi cloning site of pBPE. trpC promoter and terminator were used for expression of geneticin resistant gene, hygromycin resistant gene, and target gene in multi cloning site, respectively. Geneticinr, geneticin resistant gene; PtrpC, trpC promoter; MCS, multi cloning site; TtrpC, trpC terminator; Hygr, hygromycin resistant gene; Ampr, ampicillin resistant gene.
J. BIOSCI. BIOENG., pellet. Following the manipulation, 50 mL of protoplast suspension was plated onto YMA (5 g of peptone, 3 g of yeast extract, 3 g of malt extract, 10 g of glucose, and 20 g of agar per L) containing a final concentration of 3.8% NaCl as an osmotic stabilizer and 150 mg/mL G418 disulfate aqueous solution (Nacalai Tesque, Kyoto, Japan). YM containing a final concentration of 0.5% agar (gelling temperature 30e31 C; Nacalai Tesque), 3.8% NaCl, and 150 mg/mL G418 was overlaid on protoplasts. The plates were incubated at 30 C until colonies were visible, then growth of geneticin resistant colonies was examined on YMA plate containing 150 mg/mL G418 or without its drug. Wild type and transformants grown on YMA plate at 30 C, for 7 days. GFP fluorescence was imaged using a Typhoon 9410 imager (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire , UK) using a 488 nm laser for excitation and a 526 nm short pass (SP) filter to collect the emission. GFP expression was observed using a fluorescence microscope (Keyence, Osaka, Japan). The pBPE sequence inserted in the genome was detected by PCR using GFPsense and GFP-antisense as vector-specific primers. Growth of selected colonies were evaluated (Fig. S1). Wild type and all drug resistant strains grew better in YMA. Moreover, drug resistant strains also could grow in its medium containing G418 although growth of wild type was inhibited. From these results, it was found that six strains are able to grow stably in presence of G418. GFP expression was detected (Fig. 2A). TP6, TP7, and TP8 exhibited high signal intensity, whereas no signal was detected
FIG. 2. (A) GFP expression in the transformants. Typhoon 9410 was used for detection of GFP signal. The wild type and transformants were grown on YMA at 30 C, for 7 days. W indicates wild type and TP indicates transformants of P. purpurogenum IAM15392. (B) Localization of GFP. The wild type and TP6 were grown on PD at 30 C, for 4 days. GFP expression was confirmed by fluorescence microscopy. (a, c) Bright field images of the transformants. (b, d) GFP expression in panels a and c, respectively. (e, f) Images of wild type. (a, b, e, f) Microscope magnification 400. (c, d) Microscope magnification 200. Bars: 50 mm.
Please cite this article in press as: Kojima, R., et al., Construction of transformation system in Penicillium purpurogenum, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.08.016
VOL. xx, 2014
TECHNICAL NOTE
TABLE 1. The result of transformation in P. purpurogenum IAM15392. Amount of pBPE-GFP (mg) 5 10
Total colonies
Number of transformants
Percentage of transformants (%)a
21 24
0 3
0 12.5
3
industry and for biomass degradation. Hence, work should continue to develop an efficient transformation system and vector providing higher gene expression. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2014.08.016.
a Percentage of transformants was regarded as number of GFP fluorescence strains/total colonies.
References from TP1, TP2, and TP13. Three transformants were thus obtained. The location of GFP in T6 was observed using fluorescence microscopy (Fig. 2B) and determined to be accumulated in the hypha. Ideally, GFP mutants should be grown in medium in which no pigment is produced because pigments produced by P. purpurogenum IAM15392 cause intrinsic fluorescence. These results showed that the trpC promoter and terminator from Aspergillus nidulans can be used in P. purpurogenum. Both geneticin and hygromycin worked as selectable markers, but we found that geneticin has higher selective pressure than hygromycin (data not shown). Furthermore, although many false positive colonies were obtained using PDA and PP-O production medium containing hygromycin B (Wako, Osaka, Japan) (data not shown), few false positives (data not shown) were obtained using YMA containing hygromycin B, suggesting that the selective pressure of this drug depends on the medium. YMA medium was better for obtaining transformants of P. purpurogenum IAM15392. Transformation data are summarized in Table 1. Either 5 mg or 10 mg of pBPE-GFP were used to transform the fungus. In both cases the total number of colonies were almost the same, but the ratio of transformants to it was vastly different. When 5 mg of pBPE-GFP was used, the rate was 0%, whereas the rate was 12.5% when 10 mg of the vector was used. Transformation requires favorable conditions. For example, the number of stable colonies depends on quantity of plasmid in A. nidulans (15). Moreover, it has been reported that the highest co-transformation efficiency (7%) was observed in Penicillium griseoroseum when the ratio of two plasmids was 3:6; when equal amounts of the two plasmids were used, co-transformation efficiency decreased (16). These results suggest that 10 mg provides more transformants than 5 mg in P. purpurogenum. However, only three transformants among 24 geneticin resistant colonies obtained in 10 mg. It is possible that this is because geneticin resistance in the host was newly induced by spontaneous mutation or that geneticin resistant strains with no fluorescence have not been introduced with GFP gene but with the drug resistant gene only although a strain having both GFP genes and geneticin resistant gene is preferred. In summary, although we could construct a transformation system for P. purpurogenum, improvements to this method are required to increase transformation efficiency. In future, genetic modification of P. purpurogenum may increase its utility in the food
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Please cite this article in press as: Kojima, R., et al., Construction of transformation system in Penicillium purpurogenum, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.08.016
