An engineered mutant of Saccharomyces cerevisiae affected in biotin biosynthesis has been isolated. This mutant allowed the characterization of a bio cluster (BIO3-4-5). We demonstrate that BIO3 (YNR058w) and BIO4 (YNR057c) encode, respectively, a 7, 8-diaminopelargonic acid aminotransferase and a dethiobiotin synthase, involved in the biotin biosynthesis pathway. A novel gene, BIO5 (YNR056c), is present immediately downstream from BIO4. This gene encodes Bio5p, a protein with 11 putative transmembrane regions. Uptake experiments performed with labeled 7-keto 8-aminopelargonic acid indicate that Bio5p is responsible for transport into the cell of 7-keto 8-aminopelargonic acid.