The proportion of mycolic acid containing trans-substituents at the proximal position of the meromycolate chain is an important determinant of fluidity of the mycobacterial cell wall and is directly related to the sensitivity of mycobacterial species to hydrophobic antibiotics. MMAS-1, an enzyme encoded in the gene cluster responsible for the biosynthesis of methoxymycolates, was overexpressed in Mycobacterium tuberculosis and shown to result in the overproduction of trans-cyclopropane and trans-olefin- containing oxygenated mycolic acids. MMAS-1 converted a cis-olefin into a trans-olefin with concomitant introduction of an allylic methyl branch in a precursor to both the methoxy and ketone-containing mycolic acids. In addition to an increase in the amount of trans-mycolate, MMAS-1 expression resulted in a substantial increase in the amount of ketomycolate produced relative to methoxymycolate. Thus MMAS-1 may act at a complex branch point where expression of this enzyme directly affects the cis- to trans-ratio and indirectly affects the keto to methoxy ratio. Overexpression of MMAS-1 resulted in a substantially slower growth rate at moderately elevated temperature, decreased thermal stability of the cell wall as measured by differential scanning calorimetry, and an increased permeability to chenodeoxycholate. These results provide experimental evidence for the intermediacy of trans-olefinic mycolate precursors in trans-cyclopropane formation and suggest that increasing the proportion of the polar ketomycolate subclass may exert a significant fluidizing effect on the cell wall.