Abstract
In Hypocrea jecorina (anamorph: Trichoderma reesei) multiple gene deletions are limited by the number of readily available selection markers. We have therefore constructed a blaster cassette which enables successive gene knock-outs in H. jecorina. This 3.5 kb pyr4 blaster cassette contains the H. jecorina pyr4 marker gene encoding orotidine-5′-monophosphate (OMP) decarboxylase flanked by two direct repeats of the Streptoalloteichus hindustanus bleomycin gene (Sh ble), which facilitate the excision of the blaster cassette by homologous recombination after each round of deletion. Functionality of this pyr4 blaster cassette was demonstrated by deletion of the glk1 encoding glucokinase and hxk1 encoding hexokinase. 1.4–1.8 kb of the non-coding flanking regions of both target genes were cloned into the respective blaster cassettes and transformation of a pyr4 negative H. jecorina strain with the two cassettes resulted in 10–13% of the transformants in the deletion of one of the two kinase genes. For excision of the pyr4 blaster cassettes, Δglk1 strains were selected for growth in the presence of 5-fluoroorotic acid. Recombination between the two Sh ble elements resulted in uridine auxotrophic strains which retained their respective glucokinase negative phenotype. Subsequent transformation of one of these auxotrophic Δglk1 strains with the hexokinase blaster cassette resulted in pyr4 prototrophic strains deleted in both glk1 and hxk1. Δglk1 strains showed reduced growth on d-glucose and d-fructose whereas Δhxkl strains showed reduced compact growth on d-glucose but were unable to grow on d-fructose as carbon source. The double Δglk1Δhxk1 deletion strain was completely unable to grow on either d-glucose or d-fructose.
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References
Alani E, Cao L, Kleckner N (1987) A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics 116:541–545
Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346
Chiang C, Knight SG (1961) L-Arabinose metabolism by cell-free extracts of Penicillium chrysogenum. Biochim Biophys Acta 46:271–278
d’Enfert C (1996) Selection of multiple disruption events in Aspergillus fumigatus using the orotidine-5′-decarboxylase gene, pyrG, as a unique transformation marker. Curr Genet 30:76–82
Fekete E, Karaffa L, Sandor E, Banyai I, Seiboth B, Gyemant G, Sepsi A, Szentirmai A, Kubicek CP (2004) The alternative D-galactose degrading pathway of Aspergillus nidulans proceeds via L-sorbose. Arch Microbiol 181:35–44
Flipphi M, van de Vondervoort PJ, Ruijter GJ, Visser J, Arst HN Jr, Felenbok B (2003) Onset of carbon catabolite repression in Aspergillus nidulans. Parallel involvement of hexokinase and glucokinase in sugar signaling. J Biol Chem 278:11849–11857
Fonzi WA, Irwin MY (1993) Isogenic strain construction and gene mapping in Candida albicans. Genetics 134:717–728
Gruber F, Visser J, Kubicek CP, de Graaf LH (1990a) Cloning of the Trichoderma reesei pyrG-gene and its use as a homologous marker for a high-frequency transformation system. Curr Genet 18:447–451
Gruber F, Visser J, Kubicek CP, de Graaff LH (1990b) The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-negative mutant strain. Curr Genet 18:71–76
Ilmen M, Thrane C, Penttila M (1996) The glucose repressor gene cre1 of Trichoderma: isolation and expression of a full-length and a truncated mutant form. Mol Gen Genet 251:451–460
Krappmann S, Braus GH (2003) Deletion of Aspergillus nidulans aroC using a novel blaster module that combines ET cloning and marker rescue. Mol Genet Genomics 268:675–683
Kubicek CP, Harman GE (eds) (1998) Trichoderma and Gliocladium. Taylor and Francis Ltd, London
Kuhls K, Lieckfeldt E, Samuels GJ, Kovacs W, Meyer W, Petrini O, Gams W, Borner T, Kubicek CP (1996) Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina. Proc Natl Acad Sci USA 93:7755–7760
Mach RL (2004) Transformation and gene manipulation in filamentous fungi: an overview. In: Arora DK (ed) Handbook of Fungal Biotechnology, 2nd edn. Marcel Dekker, New York, pp 109–119
Mandels MM, Andreotti RE (1978) The cellulose to cellulase fermentation. Proc Biochem 13:6–13
Mio T, Yabe T, Sudoh M, Satoh Y, Nakajima T, Arisawa M, Yamada-Okabe H (1996) Role of three chitin synthase genes in the growth of Candida albicans. J Bacteriol 178:2416–2419
Muhlschlegel FA, Fonzi WA (1997) PHR2 of Candida albicans encodes a functional homolog of the pH-regulated gene PHR1 with an inverted pattern of pH-dependent expression. Mol Cell Biol 17:5960–5967
Penttilä ME (1998) Heterologous protein production in Trichoderma. In: Harman GE, Kubicek CP (eds) Trichoderma and Gliocladium. Taylor and Francis Ltd., London, pp 356–383
Pla J, Gil C, Monteoliva L, Navarro-Garcia F, Sanchez M, Nombela C (1996) Understanding Candida albicans at the molecular level. Yeast 12:1677–1702
Punt PJ, Seiboth B, Weenink XO, van Zeijl C, Lenders M, Konetschny C, Ram AF, Montijn R, Kubicek CP, van den Hondel CA (2001) Identification and characterization of a family of secretion-related small GTPase-encoding genes from the filamentous fungus Aspergillus niger: a putative SEC4 homologue is not essential for growth. Mol Microbiol 41:513–525
Roberts CF (1963) The genetic analysis of carbohydrate utilization in Aspergillus nidulans. Gen Microbiol 31:45–58
Ruijter GJ, Panneman H, van den Broeck HC, Bennett JM, Visser J (1996) Characterisation of the Aspergillus nidulans frA1 mutant: hexose phosphorylation and apparent lack of involvement of hexokinase in glucose repression. FEMS Microbiol Lett 139:223–228
Sambrook J, Russel DW (2001) Molecular cloning. A laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sanglard D, Hube B, Monod M, Odds FC, Gow NA (1997) A triple deletion of the secreted aspartyl proteinase genes SAP4,SAP5, and SAP6 of Candida albicans causes attenuated virulence. Infect Immun 65:3539–3546
Seiboth B, Hartl L, Pail M, Fekete E, Karaffa L, Kubicek CP (2004) The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on d-galactose. Mol Microbiol 51:1015–1025
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
Acknowledgements
L.H. and B.S. were supported by grant P16143 from the Austrian Science Foundation FWF and by the Hochschuljubiläumsstiftung der Stadt Wien. Sequence data were obtained from the Department of Energy Joint Genome Institute (http://www.jgi.doe.gov). The T. reesei genome sequencing project was funded by the United States Department of Energy. We thank Christian P. Kubicek for critically reading the manuscript.
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Communicated by U. Kück
Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers DQ068384 (H. jecorina glk1) and DQ068385 (H. jecorina hxk1).
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Hartl, L., Seiboth, B. Sequential gene deletions in Hypocrea jecorina using a single blaster cassette. Curr Genet 48, 204–211 (2005). https://doi.org/10.1007/s00294-005-0011-8
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DOI: https://doi.org/10.1007/s00294-005-0011-8