Clavulanic acid is produced by Streptomyces clavuligerus and is a potent inhibitor of β-lactamases, enzymes that hydrolyze and inactivate conventional β-lactam antibiotics such as penicillins and cephalosporins. It differs from all other known clavams due to its unique 5R stereochemistry, which is also responsible for its bioactivity. Clavulanic acid and the 5S clavams share a common biosynthetic pathway in S. clavuligerus and precursors leading up to the penultimate step during clavulanic acid biosynthesis also have 5S stereochemistry. As to how and when the stereochemical change from 5S to 5R takes place during production has been a long standing question, with many different hypotheses being proposed. One such hypothesis involves the product of orf12 (or Ble) from the clavulanic acid gene cluster, which resembles class A β-lactamases and has an additional N-terminal domain resembling steroid isomerases/cyclases. Previous reports from other groups have shown that Ble exhibits some in vitro β-lactam esterase activity (Acta Crystallogr D Biol Crystallogr. 2013.69:1567-79), the implications of which on clavulanic acid biosynthesis is not clear. We conducted extensive in vivo mutagenesis, expression and complementation studies to show that Ble is most likely a bonafide enzyme from the clavulanic acid biosynthetic pathway of S. clavuligerus. It is intriguing that a protein related to β-lactamases would be involved in the biosynthesis of a β-lactamase inhibitor, the implications of which will also be discussed.