Although the antifungal drug Amphotericin B (AmB) effectively kills fungi, it also exhibits toxicity toward humans. AmB is believed to act by creating channels through cell membranes, causing critical cell components to leak out, eventually killing the cell. Its activity has been shown to involve its sugar component, mycosamine. This project contributes to our long-term goal of using the natural glycosyltransferase, AmphDI, to attach alternate sugars to the drug core in search of a more potent antifungal agent with lessened toxicity. This would allow for antifungal treatment with a single drug, as opposed to current antifungal therapy, which typically relies on treatment with a combination of drugs. In this work, we investigated heterologous expression of AmphDI. The polymerase chain reaction was used to amplify the gene, amphDI, from wild-type Streptomyces nodosus; the gene was then inserted into an Escherichia coli-compatible plasmid. The positively identified construct was verified by restriction enzyme digest, and standard E. coli BL21(DE3) was then transformed with the construct to test enzyme expression. Despite testing a variety of growth conditions designed to enhance enzyme expression, no overexpression was observed. Closer scrutiny of the amphDI gene sequence revealed a significant number of codons rarely used by E. coli, which may have contributed to the lack of enzyme production. Therefore, we are using a Gram-positive bacterium, Streptomyces venezuelae, which is more closely related to the natural AmB-producing organism, to express the enzyme. We will present the results of our studies and discuss future plans.