ORIGINAL PAPER R. Radzio á U. KuÈck Ef®cient synthesis of the blood-coagulation inhibitor hirudin in the ®lamentous fungus Acremonium chrysogenum Received: 24 December 1996 / Received revision: 28 February 1997 / Accepted: 7 March 1997 Abstract The isopenicillin-N-synthetase-encoding pcbC gene from the ®lamentous fungus Acremonium chryso- genum is dierentially expressed in strains showing ei- ther a high or low cephalosporin C production. For a case study to demonstrate heterologous protein synthe- sis in A. chrysogenum, we have chosen a synthetic 195-bp gene encoding the thrombin inhibitor hirudin from the leech Hirudo medicinalis. The hirudin gene was fused with the 5¢ and 3¢ regions of the pcbC gene, resulting in four dierent expression vectors, which we named pHIR1 to pHIR4. In order to achieve secretion of the heterologous polypeptide, two out of four vectors carry, in addition, secretion signal sequences of an alkaline protease gene originating either from Fusarium sp. or from A. chrysogenum. After DNA-mediated transfor- mation of the two A. chrysogenum strains, transformants were further analysed on the transcriptional and trans- lational level. Irrespective of the vector used for transformation, all transformants show a hirudin-gene- speci®c transcript in Northern hybridizations. In further analysis, hirudin synthesis was determined with a thrombin-inhibition assay, but was detectable only in those strains carrying expression plasmids with the se- cretion signals. In this case, hirudin was secreted into the culture medium. Transformants from strains with a high cephalosporin C production showed a three- to eightfold higher expression of the hirudin gene compared to low cephalosporin-C-producing strains. The amount of re- combinant hirudin was quanti®ed further by ELISA and Western blotting, using a monoclonal antibody directed against recombinant hirudin. Finally, the time course of hirudin gene expression was investigated in a selected transformant that has hirudin activities of 8.0 ATU/ml culture medium. Northern hybridization experiments revealed the highest hirudin transcript level after 2±5 days of cultivation, showing the strongest signal after 3 days. After 4±5 days, we detected the highest hirudin activity, as was con®rmed by Western blotting. The level of het- erologous hirudin synthesis in A. chrysogenum is discus- sed in relation to other eukaryotic expression systems. Introduction Filamentous fungi are traditionally used for biotechnical applications and, therefore, the procedures for the large- scale production of pharmaceutically or technically rel- evant substances are well established. Owing to the availability of ecient systems for DNA-mediated transformation of many fungal species (for review, see Lemke and Peng 1995; Finkelstein 1992; Fincham 1989), ®lamentous fungi have attracted special interest as host organisms for the production of heterologous proteins. The main feature, which makes ®lamentous fungi preferable hosts for heterologous gene expression, is their ability to perform posttranslational modi®cations correctly and subsequently to secrete the foreign protein with high eciency (for review, see Peberdy 1994; Punt et al. 1994). Moreover, many industrially important species have the GRAS (generally recognised as safe) status from the US food and drug administration because of the long-term experience in handling these strains. At present, the majority of fungal expression systems have been especially developed for Trichoderma and for various Aspergillus species because of their industrial importance (for recent reviews, see Archer 1994; Ver- does et al. 1995). Another fungal species of great biotechnical relevance is the b-lactam producer Acre- monium chrysogenum. Although it is the most important industrial producer of the antibiotic cephalosporin C, only a few attempts have been made to use this well- established fungus for the synthesis of heterologous proteins (Morita et al. 1994b, 1995). None of these systems have used a homologous promoter to drive the expression of a heterologous gene. Appl Microbiol Biotechnol (1997) 48: 58±65 Ó Springer-Verlag 1997 R. Radzio á U. KuÈck (&) Lehrstuhl fuÈr Allgemeine Botanik, Ruhr-UniversitaÈt Bochum, D-44780 Bochum, Germany Tel.: +49 234 7006212 Fax: +49 234 7094184