Eur J Appl Microbiol Biotechnol (1982) 15 : 41-46 European AIII,'~ ;~,~ Journan of n'tl,./IJIIR~U Microbiologyand Bioteehn ogy 9 Springer-Verlag. 1982 Comparison of Penicillins Produced by Inter-Species Hybrids from Penicillium chrysogenum Jozef Annd Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium Summary. From inter-species heterokaryons produced fol- lowing induced protoplast fusion between Penicillium chrysogenum and P. stoloniferum, P. patulum or P. ver- rucosum var. cyclopium, hybrid progeny with different stability and morphology could be isolated. Analysis of penicillins excreted from the hybrid progeny showed that antibiotic production was influenced both quantitatively and qualitatively. In most instances, and ex- cept for P. chrysogenum + P. stolon@rum hybrids, penicil- lin yield was adversely affected after inter-species hybridiza- tion. Investigations of the different natural penicillins produced, including pentyl-, benzyl- and heptylpenicillin, indicated that the relative amount of pentylpenicillin was considerably decreased in favour of benzyl- and/or heptylpenicillin. In addition, as observed for P. chry- sogenum + P. verrucosum var. cyclopium, genes from dif- ferent species and coding for different biochemical proper- ties could simultaneously be expressed in inter-species hybrids. Introduction The transfer of genetic material using conventional sexual or parasexual techniques has been applied for strain im- provement of industrial fungi only to a very limited ex- tent (Elander et al. 1973; Calam et al. 1976; MacDonald and Holt 1976). The recent developments in both cellular and molecular genetics, including protoplast fusion and recombinant DNA technology, raised, however, the interest for a new genetic approach in microbial breeding (Demain 1980; Hopwood and Chater 1980; Elander 1980). Useful application of genetic recombination using pro- toplast fusion is already demonstrated for strains of Cephalosporium acremonium (Hamlyn and Ball 1979). In addition, genetic recombination, especially when fusing strains from different species, could influence metabolite production or may even lead to the development of new metabolites, since in this way biochemical properties of two species may be combined into a single genome. Although in filamentous fungi hybridization can be obtained following inter-species protoplast fusion (Ann6 1977; Peberdy 1979; Ferenczy 1981), no data are available so far concerning the influence of inter-species hybridiza- tion on antibiotic production. In this report, crosses be- tween Penicillium chrysogenum and several distantly re- lated Penicillium species are described and the penicillin production of some of their somatic hybrids are compared. Materials and Methods Strains. All mutant strains used in the experiments were obtained after U.V.-irradiation. Penicillium chrysogenum leu pab ylo was derived from the low yielding penicillin producing strain Wis. 49-2105 (ATCC 11710). Orn inoylo and orn lys were isolated from the higher yielding strain P. chrysogenum (gen-5, Gist- Brocades, Delft, The Netherlands). For P. stoloniferum MUCL 14586 (G. L. Hennebert, U.C.L., Louvain-la-Neuve, Belgium) mutant lys and for P. patulum (Rega Institute, Mycological Col- lection) mutants his and ade hyp whi were applied. Fusion experi- ments with P. lanosurn (CBS 343.61) were carried out with the auxotrophic mutant his met. ForP. nigricans (CBS 279.47) lysand met nic auxotrophs were used. Mutants isolated from P. verrucosum vat. cyclopium (CBS 123.14) were met and his and forP. cyclopium (NRRL 1888) met cys and pro were used in the fusion experiments. Taxonomical relationship between the species is extensively described by Raper and Thorn (1949). Media. Composition of the media used for maintenance and cultiva- tion of the strains and their protoplasts have been described else- where (Annd 1977). Strains were maintained on complete medium (CM). Mycelium for protoplast production was grown in a liquid production medium (PM) and selection of the heterokaryons ob- tained after protoplast fusion was carried out on minimal medium (MM) containing 0.6 M sucrose. The total number of protoplasts 0171-1741/82/0015/0041/$01.20