LAUSR

&NA; AccD6 is an important component of acetyl‐CoA/propionyl‐CoA carboxylase, which acts as a key role in mycolic acid synthesis and short chain fatty acyl‐coenzyme A metabolism. In this study, we demonstrated that AccD6 of Mycobacterium smegmatis associates with AccA3 (&agr; subunit of acetyl‐CoA carboxylase, MSMEG_1807) and AccE (&egr; subunit, MSMEG_1812) to form the acetyl‐CoA (propionyl‐CoA) carboxylase. Results showed that the MSMEG_4331 subunit is a regulator that interacts with the promoter region of accD6 to inhibit its transcription. Transcription of accD6 was reduced by 50% in the mutant M. smegmatis strain overexpressing MSMEG_4331. Moreover, the activity of AccD6 was inhibited by acylation (such as acetylation and propionylation). These results demonstrate that AccD6 of M. smegmatis is regulated at both the transcriptional and post‐translational levels. Our findings highlight the novel regulatory mechanism underlying mycolic acid biosynthesis in mycobacteria.