Appl Microbio| Biotechnol (1991) 34:765-771 017575989100064Z Applied Microbiology Biotechnology © Springer-Verlag 1991 Construction and physiological characterization of glyceraldehyde-3-phosphate dehydrogenase overproducing transformants of Asper[tillus nidulans P. P. F. Hanegraaf 1., P. J. Punt 2, C. A. M. J. J. van den Hondel 2, J. Dekker 1, W. Yap 1, H. W. van Verseveld 1, and A. H. Stouthamer ~ 1 Department of Biology, Free University, Amsterdam, The Netherlands 2 TNO Medical Biological Laboratory, Rijswijk, The Netherlands Received 27 July 1990/Accepted 13 November 1990 Summary. The construction and characterization of glyceraldehyde-3-phosphate-dehydrogenase (GPD) overproducing transformants of Aspergillus nidulans and their behaviour in acetate-limited continuous cul- tures and glucose-grown batch cultures are described. The A. nidulans acetamidase deletion strain MH1277 was transformed with the homologous gpdA gene on a vector with the homologous acetamidase-gene (amdS) as a selection marker. Transformant A1 contains about nine integrated copies of the gpdA gene, and shows a proportional gene-dosage GPD production of about 22% of the total soluble cell protein. Compared to the wild-type MH1277, A1 has higher growth yields and reaches higher specific growth rates on both acetate and glucose, which could be due to the key position of GPD in glycolysis and gluconeogenesis. Introduction Generally, filamentous fungi can utilize a large variety of substrates. The secrete a wide range of extracellular enzymes degrading many natural polymers (see McCul- lough et al. 1986). Filamentous fungi are frequently used in industry for the production of enzymes (e.g. proteases), primary metabolites (e.g. organic acids) and secondary metabolites (e.g. antibiotics). Furthermore, the number of filamentous fungi for which successful transformation procedures are described and thus are accessible to modern genetic manipulation techniques has increased rapidly (for a review see Fincham 1989). When used as hosts for the production of homologous or heterologous proteins, high expression levels can be reached (Finkelstein 1987). Of special interest are re- cent reports on the production of correctly processed and biologically active mammalian proteins, secreted * Present address: Biological Laboratory, Department of Micro- biology, Vrije Universiteit, de Boelelaan 1087, 1081 HV Amster- dam, The Netherlands Offprint requests to: P. P. F. Hanegraaf into the medium in relatively large amounts in compar- ison to other hosts (for a review see Saunders et al. 1989). Detailed studies of the physiology and bioenergetics of protein production of fungi have not been performed so far. However, a model which describes the physiol- ogical consequences of overproduction of intra- and extracellular proteins in terms of energy costs and sub- strate flows, will be useful in optimization of protein production. As a model system for research on intracel- lular protein production we have chosen overproduc- tion of the homologous glycolytic enzyme gly- ceraldehyde-3-phosphate-dehydrogenase (GPD; EC 1.2.1.12) in Aspergillus nidulans. Most of the genetic and physiological work on filamentous fungi so far has been carried out with A. nidulans. The gpdA gene has recently been cloned, it has a strong promotor and the GPD enzyme is already present in relatively large quan- tities in wild-type cells (about 2.5% of the total soluble cell protein) (Punt et al. 1988). This paper reports on the construction of transfor- mants of A. nidulans which overproduce the GPD pro- tein. Growth parameters of the wild-type and the over- producing transformant A1 (with nine copies of the gpdA gene) in acetate-limited continuous culture and in regulated batch cultures on glucose, are compared. Materials and methods Strains and plasmids. Escherichia coli K-12 JM109 (Yanisch-Per- ron et al. 1985) was used for the construction and propagation of vectors. A. nidulans strain MH1277 (bi.41, amdS320, amdI18, amdA7, niiA4) (Hynes et al. 1983) was used as the recipient for transformation. Plasmid pGW325, containing the amdS gene from A. nidulans was described by Wernars et al. (1986). Plasmid pAN5-22 containing the gpdA gene from A. nidulans was de- scribed by Punt et al. (1988). Recombinant DN`4 techniques..4, nidulans MH1277 was trans- formed essentially as described by Yeiton et al. (1984), Selection was carried out as described by Wernars et al. (1985) and isolation of genomic DNA from fungal cells for Southern blotting followed Yelton et al. (1984). Southern hybridization was carried out ac-