The acvA gene from Aspergillus nidulans encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase was overexpressed by replacing the wild-type acvA promoter with the ethanol dehydrogenase promoter, alcAp, from A. nidulans. The expression level of alcAp was determined using a strain in which the reporter gene, lacZ, is under the control of alcAp, and was found to be up to 100 times greater than that from the acvA promoter when induced in fermentation conditions. Penicillin yields were found to increase by as much as 30-fold when the acvA gene was overexpressed. Glucose, which strongly represses transcription from alcAp, also repressed penicillin biosynthesis in the overexpression strain. These results prove that ACV synthetase is a rate limiting enzyme for penicillin production in A. nidulans.