IsolationofTrichodermareeseiPyrGnegativeMutantbyUV.doc

精品論文推薦isolation of trichoderma reesei pyrg negative mutant by uvmutagenesis and its application in transformation1long hao, wang tianhong*, zhang yingkuanstate key laboratory of microbial technology, shandong university, jinan, p. r. china (250100)e-mail：abstracttwo uridine auxotrophic mutants of trichoderma reesei were isolated by resistance to 5-fluorooroticacid after uv mutagenesis. one mutant, called m23, was complemented with the aspergillus niger pyrg gene carried by plasmid pab4-1. mutated pyrg gene of m23 was cloned and dna sequencing analysis indicated that a cytosine inserted into the 934-939 oligo dc position of the pyrg coding region, resulting in a frameshift mutation. transformation efficiency was approximately 200-300 transformants per g 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 co-transformed with plasmid pab4-1 attained transformation efficiency of 71.8% versus21.6% 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 system1. introductionthe filamentous fungus trichoderma reesei is widely used to produce extracellular cellulases and hemicellulases applied in the textile, pulp and food industry1. it is also known for its enormous protein secretion capacity, such as cellobiohydrolase i with production up to 40 g/l. recently, attention has been focused on development of t. reesei as a host for heterologous proteins production especially because its more animal-like glycosylation process2.development of efficient transformation system is necessary for molecular breeding and direct artificial modification of 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 could make the host use some unusualcarbon or nitrogen sources3. compared with the other methods, auxotrophic complementarygenetic transformation system is proved to be more efficient. gruber et al. has isolated onepyrg-negative mutant tu-6 from t. reesei qm9414 and proved the relatively high transformation efficiency of about 700-800 transformants per g with pyrg gene from aspergillus niger4.theorotidine-5-phosphatedecarboxylaseororotidine-5-monophosphate(omp) decarboxylase coded by pyrg gene is one of the key enzymes in uridine synthesis process. the pyrg deficient strain could only grow on medium containing uridine (uracil), which obviously reduced the background growth in transformation experimrnts. 5-fluoroorotic acid (5-foa) is used to obtain pyrg-negative mutant, as it will be converted into a toxic intermediate5-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 casstte in yeast and filamentous fungi5.in this study, a protease-deficient mutant strain trichoderma reesei m3, which was derived1supported by the grants from the national natural science foundation of china (no. 30470052), the national basic research program (973) of china (no. 2003cb716006 and 2004cb719702) and the natural science research foundation for the doctoral program of higher education ministry of china (no. 20040422042).-6-from a glucose-derepressed mutant strain t. reesei rut c30, was used as original strain to mutagenize6. taking t. reesei as a host to industrially produce homologous and heterologous proteins should be a good candidate as relative low price of carbon source used in fermentation. an easy and efficient transformation system based on pyrg-negative mutant strain m23 obtained by uv mutagenesis was developed. the experimental results substantiated that pyrgtransformation system had an advantage of high efficiency and low background.2. experimental2.1 strains and plasmidsthe protease-deficient strain t. reesei m3 was obtained previously 3. the plasmid pab4-1 was gifted by van den hondel7. the plasmid pan7-1 with hygromycin phosphotransferase gene (hph) from escherichia coli was kindly supplied by dr. p. j. punt 8. the plasmid pucvhb containing the gene of vitreoscilla hemoglobin (vgb) was obtained from dr. g. m tang9.2.2 cultures and growth conditionst. reesei strain m3 was maitained on pda slant at 30. minimal medium(mm) described by mandels and andreotti10 with 1.87g/l uridine and 1.5g/l 5-foa (sangon, shanghai, p.r.c.) added was used in screening the auxotrophic mutants. selective medium used in transformationwas minimal medium harboring 1m d-sorbitol without peptone or with 150mg/l hygromycin bwhen necessary.2.3 mutagenesis and uridine auxotrophs isolationthe spores of t. reesei m3 were collected from pda slant and suspended in sterile water. the spores were placed under a uv lamp (25w) at a distance of 45 cm and were irradiated for different periods of time. then the spores were spread on mm plates. the plates were incubated for 4 d at 30, and the numbers of colonies were counted for the analysis of survival rates11.in order to generate auxotrophic mutants, the spores were exposed to uv for 130 s. after theirradiation, 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 auxotrophs12.2.4 transformation and selection of stable transformantsprotoplast preparation and transformation of t. reesei c30m3 were carried out as described by penttil et al. 13. to identify orotidine-5-phosphate decarboxylase deficient strains，theuridine auxotrophs were transformed with plasmid pab4-1 and positive transforments survived were obtained on the medium without uridine.about 4g of plasmid pucvhb and 2g of plasmid with pab4-1 or pan7-1 were mixed up to co-transform protoplasts derived from m23 or m3 (aproximate106/ml).to obtain stable transformants, conidia were collected, diluted, and plated on mm plates with0.1% tritonx-100. after about 48 h, colonies were selected and subcultured on pda slants. after that, conidia were plated on pda lacking uridine or uracil for 2 successive generations to test the stability of the transformants.2.5 pcr and southern blot analysisafter colonies had been isolated from selective medium, pcr method combined withsouthern blot analysis were performed to identify the true transformants with vgb gene.pcr amplification with primers vhb-s (5-ttagaccagcaa accattaacatca-3) and vhb-a (5-tgagcgtacaaatctgcttcca-3) was used to isolate transformants with vgb gene inserted into the genomic dna. a rapid dna extraction method according to cassago and panepucci14 was used with some modifications. the pcr procedure was as follows: an initialdenaturation at 94 for 5 min followed by 30 cycles of denaturation at 94 for 1 min, primersannealing at 61 for 30 s, and elongation at 72 for 40 s. the final elongation step was 10 minat 72. the pcr products were analyzed by gel electrophoresis.chromosomal dna (about 5.0 g) from one t. reesei transformant was digested overnight with ecori, hindiii, ecori and hindiii (takara, dalian, china), separated on a 0.7% agarose gel, and transferred to hybond-n+filter (amersham, piscataway, usa). the vgb gene was fluorescein-labelled using an 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 genepcr amplification of the mutant pyrg gene was carried out with primers zyk7s(5-gcattgaatcgccttctccgcta-3) and zyk7a (5-aaatgaaaaatgcccgagggtgata-3), using high-fidelity pfu dna polymerase (sangon, shanghai, p.r.c.). the pcr was performed under the following conditions: an initial denaturation at 94 for 5 min followed by 30 cycles of denaturation at 94 for 1 min, primer annealing at 65 for 1 min, andelongation at 72 for 2 min. the final elongation step was 10 min at 72. the pcr product of1857 bp was purified by e.z.n.a gel extraction kit (omega bio-tek, jinan, p.r.c) and ligated into the cloning vector pgem-t (promega). the dna sequencing was performed with three primers zyk7s, 7a and 8s2 (5-cggcaaggcgtcggtggc-3) (invitrogen, shanghai, p.r.c.).3. results and discussion3.1 survival rates after uv irradiationafter exposed to uv irradiation for different periods of time (0 s, 20 s, 40 s, 60 s, 80 s, 100 s,120 s), the spores were spread on mm plates. survival curve was drawn following the count of the colonies (fig. 1). the results indicated that the survival rates declined with the extending of the uv irradiation time, and after exposure to uv for 120 s, the survival rate was less than 20%. we decided to extend the exposure time to 130 s in mutagenesis in order to make the survival ratedown to 10%15.100survival rate(%)806040200020406080 100 120 140time(s)fig. 1. survival curve of t. reesei c30m3 strain after exposed to uv light3.2 selection of the uridine auxotrophs32 colonies which could grow on minimal medium with uridine and 5-foa after uv mutagenesis were isolated and transferred to pda medium in order to collect the spores. then the spores of these strains were inoculated on both mm and mm with uridine medium to identify theuridine 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 mediumsleft :medium without uridine; right: medium with uridine3.3 mitotic stability of uridine auxotrophsthe spores of m23 and m24 were repeatedly inoculated on pda slants to produce spores for at least six generations. the progenies of both m23 and m24 still could not survive on the medium without uridine, identifying that characteristic of the auxotrophs were genetically stable.3.4 identification of orotidine-5-phosphate decarboxylase deficient strainafter transformed with plasmid pab4-1, t. reesei uridine auxotrophs m23 and m24 protoplasts were spreaded on uridine-free selective plates. no transformants were obtained using m24 as host strain, while transformants of m23 were visable on plates without uridine (data not showed). the transformants of m23 with pab4-1 grew as well as m3 on mm medium without uridine. this result indicates that t. reesei m23 is an orotidine-5-phosphate decarboxylase deficient mutant and this auxotroph can be used in transformation system with pyrg selective marker.3.5 transformation of the orotidine-5-phosphate decarboxylase deficient straintaking m23 as host strain, the transformation efficiency was approximately 200-300 colonies per g plasmid dna in pyrg transformation system. further pcr analysis revealed that nearly all of the colonies grown on uridine-free mm plates were positive transformants. compared with the drug resistance transformation system with numerous false-positive colonies (up to 50% in this study) presented, pyrg transformation system markedly decreased workload of transformants selection. plasmid pucvhb was co-transformed 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 transformants by pcr method. as to co-transformation t. reesei m23 with pab4-1, 61 vgb transformants out of85 colonies grown on the selective medium were obtained in the pyrg transformation system(table 1). gaining positive transformants became easier, more effective and timesaving using pyrg transformation systemtable1 transformation efficiency obtained from two transformation systemstransformation systerm number of colonis grownon the platesnumber of vgb transformentsidentified by pcrpercentage oftransformantspyrg856171.8%hygromycin b2807326.1%to confirm the pcr results, one of the transformants obtained from pyrg basedtransformation was elucidated by southern blot analysis using a vgb probe (fig. 3).123fig. 3 southern blot analysis of transformant with inserted vgb gene1, chromosome digested by ecori;2, chromosome digested by hindiii;3, chromosome digested by ecori and hindiiithe southern blot analysis indentified that the vgb gene had been inserted into the chromosome of transformant successfully.3.6 analysis of the mutant pyrg gene of m23the pyrg gene of m23 was cloned and sequenced (data not showed). comparing with the wild type pyrg gene of t. reesei through a blast search of the t. reesei genome sequence (/trire2/trire2.home.html), it was revealed that one additional cytosine was inserted into the 934-939 position of the pyrg coding region, resulting in frame-shift mutation and disabling the omp-decarboxylase.4. conclusionsaccording to the uridine biosynthetic pathway, the uridine auxotrophic strains could be blocked at the omp-decarboxylase (pyrg ) or omp- pyrophosphorylase level. the uridine auxotrophic of m23 obtained in our experiments was resulted from the defect of orotidine-5-phosphate decarboxylase, identified by the sequencing and plasmid complement results. in the study, the t. reesei 5-foa resistant mutants could be gained easily after uv irradiation, but only 2 out of 32 mutants were the uridine auxotrophs, and only the strain m23 could be complemented to uridine prototroph by transforming with pyrg gene. according to the analysis of pyrg gene cloned from mutants m23, there is an additional cytosine existed in the mutant gene, resulted in a frameshift mutation.in contrast with the selective markers such as drug resistance genes (e.g. hygromycin b phosphotransferase), though transformation system based on hygromycin b resistance is not restricted by genotype of the host strain, this pyrg transformation system has a great advantage of lower false-positive background in transformation experiments. in the co-transformationexperiment based on selection of hygromycin b resistance, we have found that some protoplasts could grow as tiny colonies on hygromycin b selective medium but without transformed pan7-1. existence of false-positive colonies could decrease the transformation frequency and enhance the laboratory workload for screening simultaneously. while in the pyrg transformation system, auxotroph protoplasts would not grow in the medium without uridine until they were transformed with the pyrg gene carried by pab4-1 and survive, making isolation more effective and timesaving.the transgenic strains based on pyrg transformation system are suitable as the cell factories to express heterologous proteins. using this system, glycoprotein erythropoietin gene (epo) and n-acetylglucosaminyl transferase i gene (gnt1) from human have been successfully expressed in t. reesei m23 in our lab (experimental results not published). methodoligical advance r