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Two subclusters from Streptomyces mycarofaciens, a midecamycin producer, were cloned and partially sequenced. One region was located at the 5′ end of the mid polyketide synthase (PKS) genes and contained the genes midA, midB and midC. The other region was at the 3′ end of the PKS genes and contained midK, midI and midH. Analysis of the nucleotide sequence revealed that these genes encode dTDP-glucose synthase (midA), dTDP-glucose dehydratase (midB), aminotransferase (midC), methyltransferase (midK), glycosyltransferase (midI) and an assistant gene (midH). All of these genes are involved in the biosynthesis of dTDP-D-mycaminose, the first deoxysugar of midecamycin, and in transferring the mycaminose to the midecamycin aglycone in S. mycarofaciens. Similar to gene pairs desVIII/desVII in S. venezuelae and tylMIII/tylMII in S. fradiae, the product of midH probably functions as an auxiliary protein required by the MidI protein for efficient glycosyltransfer in midecamycin biosynthesis.