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Isolation of Trichoderma reesei pyrG Negative Mutant by UV Mutagenesis and Its Application in Transformation
-... ,
CHEM. RES. CHINESE UNIVERSITIES
Available online at www.sciencedirect.com
2008,24(5),565-569 Article 10 1005-9040(2008)-05-565-05
";;'. SdenceDirect
Isolation of Trichoderma reesei pyrG Negative Mutant by UV Mutagenesis and Its Application in Transformation LONG Hao, WANG Tian-hong' and ZHANG Ying-kuan State Key Laboratory ofMicrobial Technology, Shandong University, Jinan 250100, P. R. China
Abstract Two uridine auxotrophic mutants of Trichoderma reesei were isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. One mutant, called M23, was complemented with the Aspergillus niger pyrG gene carried by plasmid pAB4-1. A mutated pyrG gene of M23 was cloned and DNA sequencing analysis indicated that a cytosine was inserted into the 934-939 oligo dC position of the pyrG coding region, resulted in a frameshift mutation. Transformation efficiency was approximately 200-300 transformants per microgram of DNA with plasmid pAB4-1. Stable transformants were obtained by monosporic culture and showed to be prototroph after successive propagation. Vitreoscilla hemoglobin expression plasmid pUCVHb was cotransformed with plasmid pAB4-1 and attained a transformation efficiency of 71.8% or of26.1% with pAN7-1. Southern blot analysis of the transformants demonstrated that plasmid pUCVHb was integrated into the chromosomal DNA. The experimental results demonstrated that the pyrG-based system was more efficient and timesaving than the conventional hygromycin B resistance-based transformation system. Keywords Trichoderma reesei; UV mutagenesis; pyrG negative strain; pyrG-based transformation system
1 Introduction The filamentous fungus Trichoderma reesei is widely used to produce extracellular cellulases and hemicellulases applied in the textile, pulp, and food industry[I,2]. It is also known for its enormous protein secretion capacity, such as cellobiohydrolase I, with a production of up to 40 gIL. Of late, attention has been focused on the development of T. reesei as a host for heterologous protein production, especially because of its more animal-like glycosylation process[3,41. Development of an efficient transformation system is necessary for molecular breeding and direct artificial modification of the metabolic pathway in filamentous fungi. There are three kinds of selective markers commonly used in filamentous fungi transformation: auxotrophic complementary genes, drug resistance genes, and genes that can make the host use some unusual carbon or nitrogen sources't'. Compared with the other systems, the auxotrophic complementary genetic transformation system has proved to be more efficient. Gruber et at. [6] has isolated one
pyrG-negative mutant TU-6 from T. reesei QM9414 and proved the relatively high transformation efficiency of about 700-800 transformants per microgram, with the pyrG gene from Aspergillus niger. Orotidine-5'-phosphate decarboxylase or orotidine-5'-monophosphate(OMP) decarboxylase, coded by the pyrG gene, is one of the key enzymes in the uridine synthesis process. The pyrG deficient strain can only grow on the medium containing uridine (uracil), which obviously reduces the background growth in the transformation experiments. As 5-fluoroorotic acid(5-FOA) is used to obtain a pyrG-negative mutant, it will be converted into a toxic intermediate 5-fluoro-UMP in prototrophic strains. This metabolic process has fulfilled a repeated transformation and gene disruption, with a single uridine auxotrophic marker, in a delicate blaster cassette in yeast and filamentous fungi'". In this study, a protease-deficient mutant strain Trichoderma reesei M3, which was derived from a glucose-derepressed mutant strain T. reesei Rut C30,
*Corresponding author. E-mail: [email protected] Received December 3, 2007; accepted January 4, 2008. Supported by the National Natural Science Foundation of China(No.30470052), the National Basic Research Program of China(Nos.2003CB716006 and 2004CB719702) and the Natural Science Research Foundation for the Doctoral Program of Education Ministry ofChina(No.20040422042). Copyright © 2008, Jilin University. Published by Elsevier Limited. All rights reserved.
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566
was used as the original strain to be mutagenizedl", T. reesei was taken as a host, to industrially produce homologous and heterologous proteins, and it should be a good candidate, with a relatively low price of carbon source used in fermentation. An easy and efficient transformation system based on the pyrGnegative mutant strain M23 obtained by UV mutagenesis was developed. The experimental results substantiated the fact that the pyrG transformation system had an advantage of high efficiency and low background.
2
Experimental
riods of time. Then the spores were spread on MM plates. The plates were incubated at 30°C for 4 d, and the number of colonies was counted, for the analysis of survi val rate[l31. To generate auxotrophic mutants, the spores were exposed to UV for 130 s. After irradiation, the spores were spread on MM plates with uridine and 5-FOA. Colonies grown on this medium were transferred to both MM and MM with uridine medium, to identify the uridine auxotrophs'P'.
2.4 Transformation and Selection of Stable Transformants Protoplast preparation and transformation of
2.1 Strain and Plasmids The protease-deficient strain T. reesei M3 was obtained previously'['. The plasmid pAB4-1 was gifted by van den Hondel[91. The plasmid pAN7-1 with hygromycin phosphotransferase gene(hph) from Escherichia coli was kindly supplied by Dr. Punt(JOJ. The plasmid pUCVHb containing the gene of Vitreoscilla hemoglobin(vgb) was obtained from Dr. Tang[llJ. The strain and plasmids used in this work are listed in Table 1. Table 1 Strain and plasmids used in this study Strain or plasmid
VoI.24
Description
Origin or reference
M3"
Derived from a glucose-derepressed mutant strain T. reesei Rut C30
[8]
pAB4-l b
Containing the A. niger pyrG auxotrophic selection marker gene
[9]
pAN7-l b
hph under the control of the PgpdA promoter
[10]
pUCVHb b
Containing the gene of Vitreoscilla hemoglobin (vgb)
[II]
a. Strain; b. plasmid.
2.2 Cultures and Growth Conditions
T. reesei strain M3 was maintained on a PDA slant at 30°C. Minimal medium(MM) described by Mandels and Andreottil", with 1.87 gIL uridine and 1.5 gIL 5-FOA(Sangon, Shanghai, China) added, was used for screening the auxotrophic mutants. Selective medium used in transformation was minimal medium, harboring I mol/L D-sorbitol without peptone or with 150 mg/L hygromycin B, when necessary. 2.3 Mutagenesis and Uridine Auxotrophs Isolation The spores of T. reesei M3 were collected from the PDA slant and suspended in sterile water. The spores were placed under a UV lamp(25 W) at a distance of 45 em and were irradiated for different pe-
T. reesei C30 M3 were carried out as described by Penttila et alysJ. To identify orotidine-5'-phosphate decarboxylase deficient strains, the uridine auxotrophs were transformed with plasmid pAB4-1, and the positive transformants that survived were obtained on the medium without uridine. About 4 I-tg of plasmid pUCVHb and 2 I-tg of plasmid pAB4-1 or pAN7-1 were mixed up to cotransform protoplasts derived from M23 or M3 6 (approximately 10 /mL). To obtain stable transformants, conidia were collected, diluted, and plated on MM plates with 0.1 % TritonX-lOO. After about 48 h, colonies were selected and subcultured on PDA slants. Following that, conidia were plated on PDA lacking uridine or uracil for two successive generations, to test the stability of the transformants.
2.5
PCR and Southern Blot Analysis
After the colonies had been isolated from the selective medium, PCR method combined with Southern blot analysis was performed to identify the true transformants with the vgb gene. PCR amplification with primers VHb-s (5'-TTAGACCAGCAA ACCATTAACATCA-3') and VHb-a(5'-TGAGCGTACAAATCTGCTTCCA-3 ') was used to isolate transformants with the vgb gene inserted into the genomic DNA. A rapid DNA extraction method according to Cassago and Panepuccil'f was used with some modifications. The PCR procedure was as follows: an initial denaturation at 94°C for 5 min was followed by 30 cycles of denaturation at 94 °C for I min. The primers were annealed at 61°C for 30 s, with elongation at 72 °C for 40 s. The final
LONG Hao et at.
No.5
elongation step was for 10 min at 72 DC. The PCR products were analyzed by gel electrophoresis. According to the ONA sequence data(GenBank accession number: M2706l), there were no EcoRI or HindIII sites within the VHB gene. Chromosomal DNA(about 5.0 ~g) from one T. reesei transformant was digested overnight with EcoRl, HindIII, EcoRl, and HindlII(TaKaRa, Dalian, China), respectively, separated on a 0.7% agarose gel, and transferred to Hybond-N'filterrAmersharn, Piscataway, USA). The vgb gene was fluorescein-labeled using the ECL random prime labeling and detection system(Amersham), and used as a probe to detect the VHb gene in the transformants.
2.6 Cloning and Sequencing of the Mutant pyrG Gene PCR amplification of the mutant pyrG gene was carried out with primers ZYK7s (5'-GCATTGAATCGCCTTCTCCGCTA-3') and ZYK7a(5'-AAATGAAAAATGCCCGAGGGTGATA3'), using high-fidelity pfu DNA polymerase(Sangon, Shanghai, China). The PCR was performed under the following conditions: an initial denaturation at 94 DC for 5 min, followed by 30 cycles of denaturation at 94 DC for 1 min. The primer annealing was at 65 DC for 1 min, and elongation at 72 DC for 3 min. The final elongation step was for 10 min at 72 DC. The PCR product of 1857 bp was purified with the help of an E.Z.N.A Gel Extraction Kit(Omega Bio-Tek, Jinan, China) and directly sequenced. The DNA sequencing was performed with three primers ZYK7s, 7a, and 8s2 (5'-CGGCAAGGCGTCGGTGGC-3')(lnvitrogen, Shanghai, China).
3
Results and Discussion
3.1 Survival Rates After UV Irradiation Having been exposed to UV irradiation for different periods oftime(O, 20, 40, 60, 80, 100 and 120 s), the spores were spread on MM plates. The survival curve was drawn based on the count of the colonies(Fig.l). The results indicated that the survival rates declined with the enlongation of the UV irradiation time, and after exposure to UV for 120 s, the survival rate was less than 20%. The authors decided to extend the exposure time to 130 s in the mutagenesis, to bring the survival rate down to 10%[17].
567 100 ~-------------, 80
20 20
Fig.I
3.2
40
60 80 Time/s
100
120 140
Survival curve of T. reesei C30 M3 strain after being exposed to UV light
Selection of the Uridine Auxotrophs
Thirty-two colonies that could grow on minimal medium with uridine and 5-FOA, after UV mutagenesis, were isolated and transferred to a PDA medium, to collect the spores. Next, the spores of these strains were inoculated on both MM and MM with uridine medium to identify the uridine auxotrophs. All the strains could survive on the medium with uridine. but only two of them, M23 and M24, could not grow on the MM medium without uridine(Fig.2). The M23 and M24 strains were primarily identified as uridine auxotrophs.
Fig.2
M23 and M24 on different mediums
Left: medium without uridine; right: medium with uridine.
3.3 Mitotic Stability of Uri dine Auxotrophs The spores of strains M23 and M24 were repeatedly inoculated on PDA slants to produce spores for at least six generations. The progenies of both strains M23 and M24 still could not survive on the medium without uridine, identifying that the characteristic of the auxotrophs was genetically stable.
3.4 Identification of Orotidine-5'-phosphate Decarboxylase Deficient Strain After having been transformed with plasmid pAB4-1, the T. reesei uridine auxotrophs, M23 and M24 protoplasts, were spread on uridine-free selective plates. No transformants were obtained with M24 as a
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CHEM. RES. CHINESE UNIVERSITIES
host strain, whereas, transfonnants of M23 were visible on plates without uridine(data not shown). The transfonnants of M23 with pAB4-1 grew as well as M3 did on MM medium without uridine. This result indicated that T. reesei M23 was an orotidine 5'-phosphate decarboxylase deficient mutant and this auxotroph could be used in the transformation system with pyrG selective marker.
3.5 Transformation of the Orotidine-5'-phosphate Decarboxylase Deficient Strain With M23 as the host strain, the transformation efficiency was approximately 200-300 colonies per microgram plasmid DNA, in the pyrG transformation system. Further PCR analysis revealed that nearly all the colonies grown on uridine-free MM plates were Table 2
Vol.24
positive transfonnants. Compared with the drug resistance transformation system with numerous false-positive colonies(up to 50% in this study) presented, the pyrG transformation system markedly decreased the workload of transfonnant selection. Plasmid pUCVHb was cotransfonned with pAN7-1 to T. reesei M3, 280 colonies grew on the hygromycin B selective medium, but only 73 of them were identified as vgb transfonnants by the PCR method. As for the cotransfonnation of T. reesei M23 with pAB4-1, 61 vgb transfonnants out of 85 colonies grown on the selective medium were obtained in the pyrG transformation system(Table 2). Gaining positive transformants became easier, more effective, and timesaving if the pyrG transformation system was used.
Comparation between transformation efficiencies obtained from two transformation systems
Transformation system
Number of colonies grown on the plates
Number of vgb transformants identified by peR
Percentage of positive transformants
pyrG Hygromycin B
85 280
61 73
71.8% 26.1 %
To confirm the PCR results, one of the transfermants obtained from pyrG based transformation was elucidated by Southern blot analysis, with vgb as a probe(Fig.3).
Fig.3 Southern blot analysis of transformant with inserted vgb gene Lanes: I. chromosome digested by EcoRl; 2. chromosome digested by HindI1l; 3. chromosome digested by EcoRl and HindI1l; 4. host M23 chromosome digested by EcoRl; 5. host M23 chromosome digested by Hindlll.
The Southern blot analysis identifies that the vgb gene has been inserted into the chromosome of host M23 successfully.
3.6 Analysis of the Mutant pyrG Gene of M23 The pyrG gene of M23 was cloned and sequenced(data not shown). Compared with the sequence of the wild-type pyrG gene of T. reesei through a blast search of the T. reesei genome sequence(http://genome.jgi-psf.org/Trire2/Trire2.home. html), one additional cytosine was inserted into the 934-939 position of the pyrG coding region, result-
ing in a frameshift mutation and decavitation of the OMP-decarboxylase.
4 Conclusions According to the uridine biosynthetic pathway, the uridine auxotrophic strains could be blocked at the OMP-decarboxylase(PyrG) or OMP-pyrophosphorylase level. The uridine auxotrophic M23 obtained in these experiments was a result of the defect of orotidine-5'-phosphate decarboxylase, identified by the sequencing and plasmid complement results. In this study, the T. reesei 5-FOA resistant mutants could be gained easily after UV irradiation, but only two out of 32 mutants were the uridine auxotrophs, and only the strain M23 could be reverted and complemented to uridine prototroph by transforming with the pyrG gene. According to the analysis of the pyrG gene cloned from mutant M23, an additional cytosine existed in the mutant gene, which resulted in a frameshift mutation. In contrast to the selective markers such as the drug resistance genes(e.g., hygromycin B phosphotransferase), although the transformation system is based on hygromycin B resistance it is not restricted by the genotype of the host strain. This pyrG transformation . system has a great advantage of lower false-positive background in transformation experiments. In the cotransfonnation experiment, based on the selection of hygromycin B resistance, the authors
No.5
LONG Hao et al.
have found that some protoplasts can grow as tiny colonies on the hygromycin B selective medium, but without transformed pAN? -1. Existence of falsepositive colonies can decrease the transformation frequency and enhance the laboratory workload for screening simultaneously. However, in the pyrG transformation system, auxotroph protoplasts will not grow in the medium without uridine until they are transformed with the pyrG gene, making isolation more effective and timesaving. As cell factories, the transgenic strains based on the pyrG transformation system are suitable for expressing heterologous proteins. Using this system, the glycoprotein erythropoietin gene(epa) and Nacetylglucosaminyl transferase I gene(gntl), from humans, have been successfully expressed in T reesei M23 in the laboratory(experimental results not published). The methodological advance reported here facilitates molecular biology study as well as strain modifications of the industrially important filamentous fungi.
569
knowledge Li Xian and Zhang Guang-tao for their help in the artwork. They also thank Dr. van den Handel, Punt P J, and Tang G. M for providing plasmids pAB4-1, pAN7-1, and pUCVHb. References [1] Wang T. H., Liu T., Wu Z. H., et al., Acta Biochim. Biophys. Sin., 2004,36,667 [2] Wang T. H., Principle and Technique ofMicrobial Molecular Breed-
ing, 1st Ed., Chemical Industry Press, Beijing, 2005, 288 [3] Maras M., van Die 1., Contreras R., et al., Glycoconj. J, 1999, 16,99 [4] Punt P. J., van Biezen N., ConesaA., et al., Trends. Biotechnol., 2002,
20,200 [5] Ruiz-Diez B., J Appl. Microbiol., 2002, 92, 189 [6] Gruber E., Visser j., Kubicek C. P., et al., Curro Genet., 1990, 18,71 [7] Hartl L., Seiboth B., Curro Genet., 2005, 48, 204 [8] Wang T. H., Liu T., Liu S. L., et al., Mycosystema, 2004, 23, 248 [9] van Hartingsveldt
w., Mattern L E., van Zeijl
C. M., et al., Molec.
Gen. Genet., 1987, 206, 71 [10] Punt P 1., Oliver R. P, Dingemanse M. A., et al., Gene, 1987, 56,
117 [II]
Liu L., Liu J., Qiu R. X., et al., Lett. Appl. Microbiol., 2003,36,358
[12] Mandels M. M., Andreotti R. E., Proc. Biochem., 1978,13,6 [13] Liu Z. B., Jeenes D. J., Areher D. B., J Microbiol., 2001, 20, 15 [14] Verdoes J. C; Punt P. 1., van der Berg P., et al., Gene, 1994, 146, 159
Acknowledgments Sequence data were obtained from the Department of Energy Joint Genome Institute (http://wwwjgLdoe.gov). The authors wish to ac-
[15] Penttila M., Nevalainen H., Ratto M., et al., Gene, 1987, 61, 155 [16] Cassago A., Panepucci R. A., Tortella A. M., et aI., BMC Microbiol., 2002,2,14 [17] Hashimoto S., Ogura M., Aritomi K., et al., Appl. Environ. Micro-
biol., 2005, 71,312
CHEM. RES. CHINESE UNIVERSITIES
Available online at www.sciencedirect.com
2008,24(5),565-569 Article 10 1005-9040(2008)-05-565-05
";;'. SdenceDirect
Isolation of Trichoderma reesei pyrG Negative Mutant by UV Mutagenesis and Its Application in Transformation LONG Hao, WANG Tian-hong' and ZHANG Ying-kuan State Key Laboratory ofMicrobial Technology, Shandong University, Jinan 250100, P. R. China
Abstract Two uridine auxotrophic mutants of Trichoderma reesei were isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. One mutant, called M23, was complemented with the Aspergillus niger pyrG gene carried by plasmid pAB4-1. A mutated pyrG gene of M23 was cloned and DNA sequencing analysis indicated that a cytosine was inserted into the 934-939 oligo dC position of the pyrG coding region, resulted in a frameshift mutation. Transformation efficiency was approximately 200-300 transformants per microgram of DNA with plasmid pAB4-1. Stable transformants were obtained by monosporic culture and showed to be prototroph after successive propagation. Vitreoscilla hemoglobin expression plasmid pUCVHb was cotransformed with plasmid pAB4-1 and attained a transformation efficiency of 71.8% or of26.1% with pAN7-1. Southern blot analysis of the transformants demonstrated that plasmid pUCVHb was integrated into the chromosomal DNA. The experimental results demonstrated that the pyrG-based system was more efficient and timesaving than the conventional hygromycin B resistance-based transformation system. Keywords Trichoderma reesei; UV mutagenesis; pyrG negative strain; pyrG-based transformation system
1 Introduction The filamentous fungus Trichoderma reesei is widely used to produce extracellular cellulases and hemicellulases applied in the textile, pulp, and food industry[I,2]. It is also known for its enormous protein secretion capacity, such as cellobiohydrolase I, with a production of up to 40 gIL. Of late, attention has been focused on the development of T. reesei as a host for heterologous protein production, especially because of its more animal-like glycosylation process[3,41. Development of an efficient transformation system is necessary for molecular breeding and direct artificial modification of the metabolic pathway in filamentous fungi. There are three kinds of selective markers commonly used in filamentous fungi transformation: auxotrophic complementary genes, drug resistance genes, and genes that can make the host use some unusual carbon or nitrogen sources't'. Compared with the other systems, the auxotrophic complementary genetic transformation system has proved to be more efficient. Gruber et at. [6] has isolated one
pyrG-negative mutant TU-6 from T. reesei QM9414 and proved the relatively high transformation efficiency of about 700-800 transformants per microgram, with the pyrG gene from Aspergillus niger. Orotidine-5'-phosphate decarboxylase or orotidine-5'-monophosphate(OMP) decarboxylase, coded by the pyrG gene, is one of the key enzymes in the uridine synthesis process. The pyrG deficient strain can only grow on the medium containing uridine (uracil), which obviously reduces the background growth in the transformation experiments. As 5-fluoroorotic acid(5-FOA) is used to obtain a pyrG-negative mutant, it will be converted into a toxic intermediate 5-fluoro-UMP in prototrophic strains. This metabolic process has fulfilled a repeated transformation and gene disruption, with a single uridine auxotrophic marker, in a delicate blaster cassette in yeast and filamentous fungi'". In this study, a protease-deficient mutant strain Trichoderma reesei M3, which was derived from a glucose-derepressed mutant strain T. reesei Rut C30,
*Corresponding author. E-mail: [email protected] Received December 3, 2007; accepted January 4, 2008. Supported by the National Natural Science Foundation of China(No.30470052), the National Basic Research Program of China(Nos.2003CB716006 and 2004CB719702) and the Natural Science Research Foundation for the Doctoral Program of Education Ministry ofChina(No.20040422042). Copyright © 2008, Jilin University. Published by Elsevier Limited. All rights reserved.
CHEM. RES. CHINESE UNIVERSITIES
566
was used as the original strain to be mutagenizedl", T. reesei was taken as a host, to industrially produce homologous and heterologous proteins, and it should be a good candidate, with a relatively low price of carbon source used in fermentation. An easy and efficient transformation system based on the pyrGnegative mutant strain M23 obtained by UV mutagenesis was developed. The experimental results substantiated the fact that the pyrG transformation system had an advantage of high efficiency and low background.
2
Experimental
riods of time. Then the spores were spread on MM plates. The plates were incubated at 30°C for 4 d, and the number of colonies was counted, for the analysis of survi val rate[l31. To generate auxotrophic mutants, the spores were exposed to UV for 130 s. After irradiation, the spores were spread on MM plates with uridine and 5-FOA. Colonies grown on this medium were transferred to both MM and MM with uridine medium, to identify the uridine auxotrophs'P'.
2.4 Transformation and Selection of Stable Transformants Protoplast preparation and transformation of
2.1 Strain and Plasmids The protease-deficient strain T. reesei M3 was obtained previously'['. The plasmid pAB4-1 was gifted by van den Hondel[91. The plasmid pAN7-1 with hygromycin phosphotransferase gene(hph) from Escherichia coli was kindly supplied by Dr. Punt(JOJ. The plasmid pUCVHb containing the gene of Vitreoscilla hemoglobin(vgb) was obtained from Dr. Tang[llJ. The strain and plasmids used in this work are listed in Table 1. Table 1 Strain and plasmids used in this study Strain or plasmid
VoI.24
Description
Origin or reference
M3"
Derived from a glucose-derepressed mutant strain T. reesei Rut C30
[8]
pAB4-l b
Containing the A. niger pyrG auxotrophic selection marker gene
[9]
pAN7-l b
hph under the control of the PgpdA promoter
[10]
pUCVHb b
Containing the gene of Vitreoscilla hemoglobin (vgb)
[II]
a. Strain; b. plasmid.
2.2 Cultures and Growth Conditions
T. reesei strain M3 was maintained on a PDA slant at 30°C. Minimal medium(MM) described by Mandels and Andreottil", with 1.87 gIL uridine and 1.5 gIL 5-FOA(Sangon, Shanghai, China) added, was used for screening the auxotrophic mutants. Selective medium used in transformation was minimal medium, harboring I mol/L D-sorbitol without peptone or with 150 mg/L hygromycin B, when necessary. 2.3 Mutagenesis and Uridine Auxotrophs Isolation The spores of T. reesei M3 were collected from the PDA slant and suspended in sterile water. The spores were placed under a UV lamp(25 W) at a distance of 45 em and were irradiated for different pe-
T. reesei C30 M3 were carried out as described by Penttila et alysJ. To identify orotidine-5'-phosphate decarboxylase deficient strains, the uridine auxotrophs were transformed with plasmid pAB4-1, and the positive transformants that survived were obtained on the medium without uridine. About 4 I-tg of plasmid pUCVHb and 2 I-tg of plasmid pAB4-1 or pAN7-1 were mixed up to cotransform protoplasts derived from M23 or M3 6 (approximately 10 /mL). To obtain stable transformants, conidia were collected, diluted, and plated on MM plates with 0.1 % TritonX-lOO. After about 48 h, colonies were selected and subcultured on PDA slants. Following that, conidia were plated on PDA lacking uridine or uracil for two successive generations, to test the stability of the transformants.
2.5
PCR and Southern Blot Analysis
After the colonies had been isolated from the selective medium, PCR method combined with Southern blot analysis was performed to identify the true transformants with the vgb gene. PCR amplification with primers VHb-s (5'-TTAGACCAGCAA ACCATTAACATCA-3') and VHb-a(5'-TGAGCGTACAAATCTGCTTCCA-3 ') was used to isolate transformants with the vgb gene inserted into the genomic DNA. A rapid DNA extraction method according to Cassago and Panepuccil'f was used with some modifications. The PCR procedure was as follows: an initial denaturation at 94°C for 5 min was followed by 30 cycles of denaturation at 94 °C for I min. The primers were annealed at 61°C for 30 s, with elongation at 72 °C for 40 s. The final
LONG Hao et at.
No.5
elongation step was for 10 min at 72 DC. The PCR products were analyzed by gel electrophoresis. According to the ONA sequence data(GenBank accession number: M2706l), there were no EcoRI or HindIII sites within the VHB gene. Chromosomal DNA(about 5.0 ~g) from one T. reesei transformant was digested overnight with EcoRl, HindIII, EcoRl, and HindlII(TaKaRa, Dalian, China), respectively, separated on a 0.7% agarose gel, and transferred to Hybond-N'filterrAmersharn, Piscataway, USA). The vgb gene was fluorescein-labeled using the ECL random prime labeling and detection system(Amersham), and used as a probe to detect the VHb gene in the transformants.
2.6 Cloning and Sequencing of the Mutant pyrG Gene PCR amplification of the mutant pyrG gene was carried out with primers ZYK7s (5'-GCATTGAATCGCCTTCTCCGCTA-3') and ZYK7a(5'-AAATGAAAAATGCCCGAGGGTGATA3'), using high-fidelity pfu DNA polymerase(Sangon, Shanghai, China). The PCR was performed under the following conditions: an initial denaturation at 94 DC for 5 min, followed by 30 cycles of denaturation at 94 DC for 1 min. The primer annealing was at 65 DC for 1 min, and elongation at 72 DC for 3 min. The final elongation step was for 10 min at 72 DC. The PCR product of 1857 bp was purified with the help of an E.Z.N.A Gel Extraction Kit(Omega Bio-Tek, Jinan, China) and directly sequenced. The DNA sequencing was performed with three primers ZYK7s, 7a, and 8s2 (5'-CGGCAAGGCGTCGGTGGC-3')(lnvitrogen, Shanghai, China).
3
Results and Discussion
3.1 Survival Rates After UV Irradiation Having been exposed to UV irradiation for different periods oftime(O, 20, 40, 60, 80, 100 and 120 s), the spores were spread on MM plates. The survival curve was drawn based on the count of the colonies(Fig.l). The results indicated that the survival rates declined with the enlongation of the UV irradiation time, and after exposure to UV for 120 s, the survival rate was less than 20%. The authors decided to extend the exposure time to 130 s in the mutagenesis, to bring the survival rate down to 10%[17].
567 100 ~-------------, 80
20 20
Fig.I
3.2
40
60 80 Time/s
100
120 140
Survival curve of T. reesei C30 M3 strain after being exposed to UV light
Selection of the Uridine Auxotrophs
Thirty-two colonies that could grow on minimal medium with uridine and 5-FOA, after UV mutagenesis, were isolated and transferred to a PDA medium, to collect the spores. Next, the spores of these strains were inoculated on both MM and MM with uridine medium to identify the uridine auxotrophs. All the strains could survive on the medium with uridine. but only two of them, M23 and M24, could not grow on the MM medium without uridine(Fig.2). The M23 and M24 strains were primarily identified as uridine auxotrophs.
Fig.2
M23 and M24 on different mediums
Left: medium without uridine; right: medium with uridine.
3.3 Mitotic Stability of Uri dine Auxotrophs The spores of strains M23 and M24 were repeatedly inoculated on PDA slants to produce spores for at least six generations. The progenies of both strains M23 and M24 still could not survive on the medium without uridine, identifying that the characteristic of the auxotrophs was genetically stable.
3.4 Identification of Orotidine-5'-phosphate Decarboxylase Deficient Strain After having been transformed with plasmid pAB4-1, the T. reesei uridine auxotrophs, M23 and M24 protoplasts, were spread on uridine-free selective plates. No transformants were obtained with M24 as a
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CHEM. RES. CHINESE UNIVERSITIES
host strain, whereas, transfonnants of M23 were visible on plates without uridine(data not shown). The transfonnants of M23 with pAB4-1 grew as well as M3 did on MM medium without uridine. This result indicated that T. reesei M23 was an orotidine 5'-phosphate decarboxylase deficient mutant and this auxotroph could be used in the transformation system with pyrG selective marker.
3.5 Transformation of the Orotidine-5'-phosphate Decarboxylase Deficient Strain With M23 as the host strain, the transformation efficiency was approximately 200-300 colonies per microgram plasmid DNA, in the pyrG transformation system. Further PCR analysis revealed that nearly all the colonies grown on uridine-free MM plates were Table 2
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positive transfonnants. Compared with the drug resistance transformation system with numerous false-positive colonies(up to 50% in this study) presented, the pyrG transformation system markedly decreased the workload of transfonnant selection. Plasmid pUCVHb was cotransfonned with pAN7-1 to T. reesei M3, 280 colonies grew on the hygromycin B selective medium, but only 73 of them were identified as vgb transfonnants by the PCR method. As for the cotransfonnation of T. reesei M23 with pAB4-1, 61 vgb transfonnants out of 85 colonies grown on the selective medium were obtained in the pyrG transformation system(Table 2). Gaining positive transformants became easier, more effective, and timesaving if the pyrG transformation system was used.
Comparation between transformation efficiencies obtained from two transformation systems
Transformation system
Number of colonies grown on the plates
Number of vgb transformants identified by peR
Percentage of positive transformants
pyrG Hygromycin B
85 280
61 73
71.8% 26.1 %
To confirm the PCR results, one of the transfermants obtained from pyrG based transformation was elucidated by Southern blot analysis, with vgb as a probe(Fig.3).
Fig.3 Southern blot analysis of transformant with inserted vgb gene Lanes: I. chromosome digested by EcoRl; 2. chromosome digested by HindI1l; 3. chromosome digested by EcoRl and HindI1l; 4. host M23 chromosome digested by EcoRl; 5. host M23 chromosome digested by Hindlll.
The Southern blot analysis identifies that the vgb gene has been inserted into the chromosome of host M23 successfully.
3.6 Analysis of the Mutant pyrG Gene of M23 The pyrG gene of M23 was cloned and sequenced(data not shown). Compared with the sequence of the wild-type pyrG gene of T. reesei through a blast search of the T. reesei genome sequence(http://genome.jgi-psf.org/Trire2/Trire2.home. html), one additional cytosine was inserted into the 934-939 position of the pyrG coding region, result-
ing in a frameshift mutation and decavitation of the OMP-decarboxylase.
4 Conclusions According to the uridine biosynthetic pathway, the uridine auxotrophic strains could be blocked at the OMP-decarboxylase(PyrG) or OMP-pyrophosphorylase level. The uridine auxotrophic M23 obtained in these experiments was a result of the defect of orotidine-5'-phosphate decarboxylase, identified by the sequencing and plasmid complement results. In this study, the T. reesei 5-FOA resistant mutants could be gained easily after UV irradiation, but only two out of 32 mutants were the uridine auxotrophs, and only the strain M23 could be reverted and complemented to uridine prototroph by transforming with the pyrG gene. According to the analysis of the pyrG gene cloned from mutant M23, an additional cytosine existed in the mutant gene, which resulted in a frameshift mutation. In contrast to the selective markers such as the drug resistance genes(e.g., hygromycin B phosphotransferase), although the transformation system is based on hygromycin B resistance it is not restricted by the genotype of the host strain. This pyrG transformation . system has a great advantage of lower false-positive background in transformation experiments. In the cotransfonnation experiment, based on the selection of hygromycin B resistance, the authors
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have found that some protoplasts can grow as tiny colonies on the hygromycin B selective medium, but without transformed pAN? -1. Existence of falsepositive colonies can decrease the transformation frequency and enhance the laboratory workload for screening simultaneously. However, in the pyrG transformation system, auxotroph protoplasts will not grow in the medium without uridine until they are transformed with the pyrG gene, making isolation more effective and timesaving. As cell factories, the transgenic strains based on the pyrG transformation system are suitable for expressing heterologous proteins. Using this system, the glycoprotein erythropoietin gene(epa) and Nacetylglucosaminyl transferase I gene(gntl), from humans, have been successfully expressed in T reesei M23 in the laboratory(experimental results not published). The methodological advance reported here facilitates molecular biology study as well as strain modifications of the industrially important filamentous fungi.
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knowledge Li Xian and Zhang Guang-tao for their help in the artwork. They also thank Dr. van den Handel, Punt P J, and Tang G. M for providing plasmids pAB4-1, pAN7-1, and pUCVHb. References [1] Wang T. H., Liu T., Wu Z. H., et al., Acta Biochim. Biophys. Sin., 2004,36,667 [2] Wang T. H., Principle and Technique ofMicrobial Molecular Breed-
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Acknowledgments Sequence data were obtained from the Department of Energy Joint Genome Institute (http://wwwjgLdoe.gov). The authors wish to ac-
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