Filamentous fungi have the capacity to produce a battery of natural products of often unknown function, synthesized by complex metabolic pathways. Unfortunately, most of these pathways appear silent, many in intractable organisms, and their products consequently unidentified. One basic challenge is the difficulty of expressing a biosynthesis pathway for a complex natural product in a heterologous eukaryotic host. Here, we provide a proof-of concept solution to this challenge and describe how the entire penicillin biosynthesis pathway can be expressed in a heterologous host. The method takes advantage of a combination of improved yeast in vivo cloning technology, generation of polycistronic mRNA for the gene cluster under study, and an amenable and easily manipulated fungal host, i.e., Aspergillus nidulans. We achieve expression from a single promoter of the pathway genes to yield a large polycistronic mRNA by using viral 2A peptide sequences to direct successful cotranslational cleavage of pathway enzymes.