<jats:title>ABSTRACT</jats:title> <jats:p> The ratio of the major monensin analogs produced by <jats:italic>Streptomyces cinnamonensis</jats:italic> is dependent upon the relative levels of the biosynthetic precursors methylmalonyl-coenzyme A (CoA) (monensin A and monensin B) and ethylmalonyl-CoA (monensin A). The <jats:italic>meaA</jats:italic> gene of this organism was cloned and sequenced and was shown to encode a putative 74-kDa protein with significant amino acid sequence identity to methylmalonyl-CoA mutase (MCM) (40%) and isobutyryl-CoA mutase (ICM) large subunit (36%) and small subunit (52%) from the same organism. The predicted C terminus of MeaA contains structural features highly conserved in all coenzyme B <jats:sub>12</jats:sub> -dependent mutases. Plasmid-based expression of <jats:italic>meaA</jats:italic> from the <jats:italic>ermE</jats:italic> ∗ promoter in the <jats:italic>S. cinnamonensis</jats:italic> C730.1 strain resulted in a decreased ratio of monensin A to monensin B, from 1:1 to 1:3. Conversely, this ratio increased to 4:1 in a <jats:italic>meaA</jats:italic> mutant, <jats:italic>S. cinnamonensis</jats:italic> WM2 (generated from the C730.1 strain by insertional inactivation of <jats:italic>meaA</jats:italic> by using the erythromycin resistance gene). In both of these experiments, the overall monensin titers were not significantly affected. Monensin titers, however, did decrease over 90% in an <jats:italic>S. cinnamonensis</jats:italic> WD2 strain (an <jats:italic>icm meaA</jats:italic> mutant). Monensin titers in the WD2 strain were restored to at least wild-type levels by plasmid-based expression of the <jats:italic>meaA</jats:italic> gene or the <jats:italic>Amycolatopsis mediterranei mutAB</jats:italic> genes (encoding MCM). In contrast, growth of the WD2 strain in the presence of 0.8 M valine led only to a partial restoration (<25%) of monensin titers. These results demonstrate that the <jats:italic>meaA</jats:italic> gene product is significantly involved in methylmalonyl-CoA production in <jats:italic>S. cinnamonensis</jats:italic> and that under the tested conditions the presence of both MeaA and ICM is crucial for monensin production in the WD2 strain. These results also indicate that valine degradation, implicated in providing methylmalonyl-CoA precursors for many polyketide biosynthetic processes, does not do so to a significant degree for monensin biosynthesis in the WD2 mutant.