<jats:title>ABSTRACT</jats:title> <jats:p> The <jats:italic>ccr</jats:italic> gene, encoding crotonyl coenzyme A (CoA) reductase (CCR), was cloned from <jats:italic>Streptomyces cinnamonensis</jats:italic> C730.1 and shown to encode a protein with 90% amino acid sequence identity to the CCRs of <jats:italic>Streptomyces collinus</jats:italic> and <jats:italic>Streptomyces coelicolor</jats:italic> . A <jats:italic>ccr</jats:italic> -disrupted mutant, <jats:italic>S. cinnamonensis</jats:italic> L1, was constructed by inserting the <jats:italic>hyg</jats:italic> resistance gene into a unique <jats:italic>Bgl</jats:italic> II site within the <jats:italic>ccr</jats:italic> coding region. By use of the <jats:italic>ermE</jats:italic> * promoter, the <jats:italic>S. collinus ccr</jats:italic> gene was expressed from plasmids in <jats:italic>S. cinnamonensis</jats:italic> C730.1/pHL18 and L1/pHL18. CCR activity in mutant L1 was shown to decrease by more than 90% in both yeast extract-malt extract (YEME) medium and a complex fermentation medium, compared to that in wild-type C730.1. Compared to C730.1, mutants C730.1/pHL18 and L1/pHL18 exhibited a huge increase in CCR activity (14- and 13-fold, respectively) in YEME medium and a moderate increase (3.7- and 2.7-fold, respectively) in the complex fermentation medium. In the complex fermentation medium, <jats:italic>S. cinnamonensis</jats:italic> L1 produced monensins A and B in a ratio of 12:88, dramatically lower than the 50:50 ratio observed for both C730.1 and C730.1/pHL18. Plasmid (pHL18)-based expression of the <jats:italic>S. collinus ccr</jats:italic> gene in mutant L1 increased the monensin A/monensin B ratio to 42:58. Labeling experiments with [1,2- <jats:sup>13</jats:sup> C <jats:sub>2</jats:sub> ]acetate demonstrated the same levels of intact incorporation of this material into the butyrate-derived portion of monensin A in both C730.1 and mutant C730.1/pLH18 but a markedly decreased level of such incorporation in mutant L1. The addition of crotonic acid at 15 mM led to significant increases in the monensin A/monensin B ratio in C730.1 and C730.1/pHL18 but had no effect in <jats:italic>S. cinnamonensis</jats:italic> L1. These results demonstrate that CCR plays a significant role in providing butyryl-CoA for monensin A biosynthesis and is present in wild-type <jats:italic>S. cinnamonensis</jats:italic> C730.1 at a level sufficient that the availability of the appropriate substrate (crotonyl-CoA) is limiting.