A full-length cDNA clone for a novel glutathione S-transferase was isolated from Arabidopsis thaliana and characterized. The cDNA encodes a polypeptide of 218 amino acids with a calculated molecular mass of 24363 Da. The sequence was most related to the theta class within the glutathione-S-transferase superfamily of enzymes. The protein encoded by the cDNA was functionally expressed and enzymically active in Escherichia coli ; glutathione-S-transferase activity with the standard enzyme substrate 1 -chloro-2,4-dinitrobenzene was demonstrated (apparent K,, 10 mM ; apparent K,, for glutathione, 0.08 mM). The enzyme is substrate specific and did not use several electrophilic reduced-glutathione acceptor molecules for conjugation. However, it efficiently catalyzed the conversion of 13-hydroperoxy-9,11,15-octadecatrienoic acid (Km, 0.67 mM) as well as 13-hydroperoxy-9,ll -0ctadecadienoic acid (K,,, 0.79 mM) to the corresponding hydroxy derivatives with concomitant formation of oxidized glutathione. The enzyme did not use H,O, as substrate. Thus, the cloned A. thaliana enzyme functions as glutathione peroxidase and, in the plant cell, may be involved in the removal of reactive organic hydroperoxides, such as the products of lipid peroxidation. The enzyme is structurally and enzymically, however, unrelated to the selenium-containing glutathione peroxidases. Enzymic and immunoblotting data suggest that the A. thaliana enzyme is soluble and constitutively expressed in vegetative rosettes, but is under developmental control during the transition to bolting and flowering.

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The isopenicillin-N-synthetase-encoding pcbC gene from the filamentous fungus Acremonium chrysogenum is differentially expressed in strains showing either a high or low cephalosporin C production. For a case study to demonstrate heterologous protein synthesis 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 different 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 transformation of the two A. chrysogenum strains, transformants were further analysed on the transcriptional and translational level. Irrespective of the vector used for transformation, all transformants show a hirudin-gene-specific 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 secretion 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 recombinant hirudin was quantified 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 confirmed by Western blotting. The level of heterologous hirudin synthesis in A. chrysogenum is discussed in relation to other eukaryotic expression systems.

β‐lactam antibiotics are commercially and clinically important compounds that are produced by bacteria as well as by filamentous fungi. There is a great interest not only to increase the yield of microbial antibiotic production but also to generate new and highly effective antibiotics. It may be foreseen that this aim is reached by the use of in vitro recombinant technology. The biochemical as well as the physiological data which seem to be important for the understanding of β‐lactam biosynthesis is filamentous fungi are summarised. In addition, recent technical advances are mentioned which become available through molecular biology. Examples are given to demonstrate the feasability of DNA recombinant technology for biotcchnical applications by introducing novel biosynthetic pathways into fungal β‐lactam producers.

Acremonium chrysogenum, a producer of cephalosporin C, was subjected to DNA-mediated transformations using a vector without bacterial DNA sequences. Recombinant fungal strains were generated with a gel-purified DNA fragment, carrying only the mutated beta-tubulin gene from A. chrysogenum. The lack of any bacterial DNA was verified by Southern hybridization analysis and polymerase chain reaction amplifications to detect even residual DNA sequences. This procedure can be referred to as a self-cloning experiment for which less restricted working regulations are needed. Finally, the transfer of a synthetic hirudin gene by cotransformation demonstrated that any DNA molecule can be introduced into the A. chrysogenum genome without bacterial marker genes. This seems to be highly relevant for biotechnical processes in which safe recombinant producer strains are required to satisfy governmental restrictions.