Microbiology (1 999), 145, 30 1-307 Printed in Great Britain Reconstitution of a bacteriallplant polyamine biosyn t hesi s pathway i n Saccharomyces cerevisiae R. D. Klein,’ T. G. Geary,’ A. 5. Gibson,’ M. A. Favreau,’ C. A. Winterrowd,’ S. J. Upton,2 J. 5. Keithlyt3G. Z~U,~ R. L. Malmberg,4 M. P. Martinez’ and N. Yarlett’ Author for correspondence: N. Yarlett. Tel: + 1 212 346 1246. Fax: + 1 212 346 1586. e-mail : nyarlett@ fsrnail.pace.edu Pharmacia and Upjohn, Animal Health Discovery Research, Kalamazoo, MI 49007, USA Division of Biology, Kansas State University, Manhattan, KS 66506, USA Wadsworth Center, NY State Department of Health, David Axelrod Institute, Albany, NY Botany Department, University of Georgia, Athens, GA 30602-727 1, USA Pace University, Haskins Laboratories, 41 Park Row, New York, NY 10038-1 598, USA 12201 -2002, USA Polyamine synthesis in most organisms is initiated by the decarboxylation of ornithine to form putrescine via ornithine decarboxylase (ODC). Plants, some bacteria and some fungi and protozoa generate putrescine from arginine, via arginine decarboxylase (ADC) and agmatine ureohydrolase (AUH) or agmatine i m i no hydro I ase. A pol yam i ne-requ i r i ng strain of Saccharomyces cerevisiae with a mutation in the gene encoding ODC was transformed with plasmids bearing genes encoding Escherichia coli ADC and AUH. Transformants regained the ability to grow in the absence of exogenous polyamines and contained enzyme activities consistent with the presence of both prokaryotic enzymes. Similar results were obtained when a plasmid containing a gene encoding oat (Avena sativa L.) ADC was substituted for the E. coli gene. These data demonstrate the successful complementation of a yeast biosynthetic polyamine synthesis defect by genes encoding an alternative pathway found in bacteria; they also show that plant ADC can substitute for the bacterial enzyme in this pathway. The recombinant yeast provides a tool for the study of the functional properties of these enzymes and for discovery of compounds that specifically inhibit this pathway. Keywords : polyamines, arginine decarboxylase, agmatine ureohydrolase, yeast INTRODUCTION Recombinant micro-organisms have many uses in bio- technology. Of most interest to us is the utility of recombinant microbes in drug discovery (Klein & Geary, 1997). One example is the use of a recombinant strain of yeast to screen for compounds that could specifically block nematode polyamine biosynthesis by inhibiting ornithine decarboxylase (ODC; Klein et al., 1997). In this case, a strain of Saccharomyces cerevisiae mutated in the ODC locus (spel) was complemented with a cDNA encoding ODC from Haemonchus contortus; assay of test compounds in the presence or absence of exogenous polyamines provided a simple screen for ODC inhibitors. Polyamine synthesis is a legitimate chemotherapeutic target for many infectious pathogens (Marton & Pegg, 1995). Among parasitic . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . .. . . . . .. .. . . . . ,, . . . . .. .. . . . . . . . . .. .. . . . .. . . . .. .. . . , . . . .. .. . . . . . . .. . . . . . . . . . .. . . . . . .. . . . . . . . . . . .. .. . . . . . . . . Abbreviations: ADC, arginine decarboxylase; AUH, agmatine ureohydrolase; DFMA, DL-a-difluoromethylarginine; DFMO, oL-ff-difluoro- rnethylornithine; ODC, ornithine decarboxylase. organisms, the protozoa Trypanosoma cruzi (Majumdar et al., 1992) and Cryptosporidium parvum (Keithly et al., 1997) have been shown not to synthesize polyamines through ODC, but through an arginine decarboxylase (ADC) /agmatine ureohydrolase (AUH) pathway typically found in some bacteria (Tabor & Tabor, 1985; Panagiotidis et af., 1987) and plants (Slocum, 1991). To determine whether a micro-organ- ism complemented for these genes could be used to screen for ADC/AUH inhibitors, experiments were designed for the functional reconstitution of a bacterial/ plant polyamine biosynthetic pathway in yeast. METHODS General methods. DNA-modifying enzymes, media and reagents were as described previously (Klein & Roof, 1988). Nucleic acid sequence analysis was performed with an ABI 377 automated sequencer (Perkin-Elmer) according to the manufacturer’s instructions. Oligonucleotides used for PCR or sequence analysis were purchased from Genosys. PCR 0002-2842 0 1999 SGM 30 1