Abstract A 0.33-kilobase (kb) DNA fragment encoding the promoter was isolated from Zymomonas chromosomal DNA as an in-frame fusion producing functional β-galactosidase. The DNA sequence of the fragment was analyzed, and a transcriptional initiation site was found. To confer the fermentation of raffinose on Z. mobilis, two recombinant plasmids were constructed: pZER193 that contains the α-galactosidase gene of E. coli inserted immediately downstream of the isolated promoter, and pZY1 that contains the lactose permease gene of E. coli. They were introduced into the strain Z6C of Zymomonas, capable of fermenting the fructose moiety of raffinose but not the melibiose moiety. Cells of the strain carrying both plasmids could ferment melibiose to ethanol, indicating that expression of α-galactosidase and lactose permease in Z6C strain expanded the substrate spectrum of Z. mobilis for ethanol production.