A practical approach is to elucidate the mechanism in which spirotryprostatin is biosynthesized, however, the genes encoding spirotryprostatins’ biosynthetic machinery remains to be identified in the chromosomal DNA of the native organism, Aspergillus fumigatus. This fungus was theorized to produce several diketopiperazines derived from brevianamide F that is biosynthesized by a non-ribosomal peptide synthetase which employs L-proline and L-tryptophan as substrates. On this presumptive evidence, our proposed biosynthetic pathway for spirotryprostatins should include synthesis of brevianamide F along with a pathway of moderating step via an oxidation reaction that is catalyzed by cytochrome P450 monooxygenases. To understand the spirotryprostatin biosynthetic pathway with aims of comprehensive reconstitution of their assembly line, we start by constructing a plasmid-borne biosynthetic gene cluster tailored for a heterologous host, S. cerevisiae. Here, we successfully demonstrate the production of a bioactive fungal molecule in a S. cerevisiae expression system using synthetic cDNAs that has been cloned to recombinantly connect the exon region obtained by means of genome template PCR.