The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: A convenient and general selection system for filamentous fungal transformation |
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| Authors: | Shiela E Unkles Edward I Campbell Yolanda M J T de Ruiter-Jacobs Martien Broekhuijsen Janet A Macro Dirk Carrez Roland Contreras Cees A M J J van den Hondel and James R Kinghorn |
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| Institution: | (1) Plant Molecular Genetics Unit, Sir Harold Mitchell Building, University of St. Andrews, KY16 St. Andrews, Fife, Scotland;(2) TNO Medical Biological Laboratory, PO. Box 45, NL-2280AA Rijswijk, The Netherlands;(3) Laboratorium voor Molculaire Biologie, State University of Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium |
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| Abstract: | Summary The development of an efficient and homologous transformation system for Aspergillus oryzae is described. This is based on nitrate reductase (niaD) of the nitrate assimilation pathway. The niaD system offers a number of inherent advantages over many other systems and may be of general use for nitrate-utilising filamentous fungi. Transformation frequencies of up to 800 transformants per microgram DNA are observed with A. oryzae. The preponderance of integration events take place at the resident niaD locus either by gene conversion (41%), single integration (23%) or multiple tandem integration (36%). Heterologous expression of the A. oryzae niaD gene in the filamentous fungi A. nidulans, A. niger and Penicillum chrysogenum is observed. That heterologous putative niaD hybridisation signals are seen with other fungal DNAs affords the oppotunity to isolate the corresponding niaD from various fungi in order to develop homolgous transformation. Co-transformation with the introduction of the non-selected markers pyrG, tub-2, and uidA has been achieved. |
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| Keywords: | Aspergillus oryzae Homologous transformation Nitrate reductase Chlorate resistance Co-transformation |
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