Mol Gen Genet (1988) 214:562-569 © Springer-Verlag 1988 Cloning and comparative sequence analysis of the gene coding for isopenicillin N synthase in Streptomyces Dov Shiffman 1, Moshe Mevarech 1, Susan E. Jensen 2, Gerald Cohen 1, and Yair Aharonowitz 1 1 Tel Aviv University, The George S. Wise Faculty of Life Sciences, Department of Microbiology, Ramat Aviv, 69978, Israel z University of Alberta, Department of Microbiology, Edmonton, Alberta, Canada, T6G 2E9 Summary. The genes coding for isopenicillin N synthase (IPNS) in Streptomyces jumonjinensis and S. Iipmanii were isolated from recombinant phage lambda libraries using the S. clavuligerus IPNS gene as a heterologous probe. The S. jumonjinensis IPNS gene has an open reading frame cod- ing for 329 amino acids, identical in size to that of the previously cloned S. clavuligerus IPNS gene. A partial nu- cleotide sequence was also determined for the S. lipmanii IPNS gene. Comparison of the predicted amino acid se- quences of all three streptomycete IPNS proteins shows that they exhibit more than 70% similarity, close to that found in comparisons among fungal IPNS proteins and significantly greater than that found, approximately 60%, between Streptomyces and fungal IPNS proteins. We con- clude that procaryotic and eucaryotic IPNS genes are sub- groups of a single family of microbial IPNS genes. Hybrid- ization probes prepared from IPNS genes of the above streptomycete species were used to detect analogous genes in eight other strains that included both penicillin and ce- phalosporin producers and non-producers. Each producer strain responded with all three probes implying the presence of an IPNS gene. Surprisingly, several non-producer strains also responded with one or two of the probes. Our results suggest that IPNS-related genes may be more prevalent in Streptomyces than previously believed. Key words: Isopenicillin-N synthase - Streptomyces - B - lactam antibiotics - Sequence similarity - Cloning Introduction Microorganisms that produce/%lactam antibiotics are wide- spread in nature (Elander 1983). Originally these com- pounds were discovered in fungi and found to consist of two main classes, the penicillins and cephalosporins. Peni- cillins are made by several funsal species including Penicil- lium chrysogenum and Aspergillus nidulans. Cephalosporins are found in Cephalosporium acremonium. More recently fl-lactam antibiotics have been shown to be made in a much more diverse group of taxonomically unrelated species. Thus, many new fl-lactam compounds have been isolated Offprint requests to: Y. Aharonowitz from actinomycetes and in particular from the genus Strep- tomyces. Moreover, with the development of more sensitive methods for detection of/~-lactam antibiotics a variety of gram-negative bacteria, Pseudomonas, Gluconobacter, Ace- tobacter and Flavobacterium, have also been shown to make these types of metabolites (Elander 1983). Microbial//-lac- tams are conveniently classified according to their chemical structure: most important of these are the penicillins, ce- phalosporins, cephamycins, clavams, carbapenems and the monocyclic/Mactams comprising nocardicins and sulfaze- cins. In the present study our analysis is confined to the first three classes. The early steps in the biosynthesis of penicillins, cepha- losporins and cephamycins are essentially the same in eucar- yotic and procaryotic organisms (Jensen 1986). A multi- functional enzyme, ACV synthetase, mediates the initial steps in their synthesis through formation of a linear tripep- tide 6-(L-~-aminoadipyl)-L-cysteinyl-D-valine (ACV) (Banko and Demain 1987). The enzyme isopenicillin N synthase (IPNS) then catalyzes the oxidative cyclization of the tripep- tide, in the presence of Oz, Fe 2÷ and ascorbate, to form the first /?-lactam intermediate isopenicillin N (Konomi et al. 1979). IPNS has been extensively purified from two funsal species, P. chrysogenum (Ramos et al. 1985) and C. acremonium (Pang et al. 1984) and two streptomycete spe- cies, Streptomyces clavuligerus (Jensen et al. 1986) and S. Iactamdurans (Castro et al. 1988). In the case of C. acre- monium and S. clavuligerus partial amino acid sequences were made at the NH2-terminal end of the IPNS proteins and the information used to construct oligodeoxynucleotide probes for isolating the corresponding genes (Samson et al. 1985; Leskiw et al. 1988). These genes can, in turn, serve as hybridization probes to isolate heterologous IPNS genes in other related fl-lactam producing organisms. Thus, the IPNS gene of C. acremonium was used to clone the analo- gous genes ofP. chrysogenum (Carr et al. 1986) and A. nidu- lans (Ramon et al. 1987). In this paper we describe the use of a hybridization probe derived from the S. clavuligerus IPNS gene to isolate the corresponding IPNS genes from S.jumonjinensis and S. lipmanii. The protein-coding regions of these genes were sequenced and the data used to compare their predicted amino acid sequences with those of other funsal and strep- tomycete IPNS genes. We also describe the use of hybridiza- tion probes from Streptomyces IPNS genes to detect analo- gous genes in other streptomycete species.