Pactamycin, a complex aminocyclitol compound produced by Streptomyces pactum, has significant antibiotic, antitumor, and antiplasmodial activity. However, this natural product has not been developed for clinical use due to its high cytotoxicity. Modulation of its biological properties by chemical synthesis has been difficult due to its complex chemical structure. Therefore, biosynthetic-based genetic engineering appears to be a feasible alternative approach to developing new analogs of pactamycin. While the pactamycin biosynthetic gene cluster is known, many parts of the pathway remain elusive. For example, the acyltransferase gene responsible for the transfer of 6-methylsalicyclic acid to the aminocyclitol core has not yet been identified. As part of our effort to identify this gene, we used gene disruption to inactivate 1) a putative acyltransferase gene, located outside of the pactamycin gene cluster but near the pactamycin resistance gene, and 2) a putative ketoacyl-(ACP) synthase (KAS)-III gene (ptmR), located within the cluster. The results showed that PtmR is the enzyme responsible for the attachment of 6-methylsalicylic acid. We also hypothesized that pactamycin production could be increased through the modulation of global regulators. Inactivation of the global regulatory genes afsA, arpA, and phoP, which are known to affect secondary metabolism, revealed that ArpA is directly involved in the regulation of pactamycin biosynthesis. On the other hand, inactivation of afsA and phoP did not affect pactamycin production.