The deep-sea-derived microbe Streptomyces scopuliridis SCSIO ZJ46 produces desotamides A-D. Notably, desotamides A and B display antibacterial activities against pathogenic Gram-positive Streptococcus pneumoniae NCTC 7466, Staphylococcus aureus ATCC 29213, and the methicillin-resistant clinical isolate Staphylococcus epidermidis (MRSE) shhs-E1. The 39-kb desotamide biosynthetic gene cluster (dsa) has previously been identified and heterologously expressed in S. coelicolor M1152 for the purposes of assigning dsa gene functions. In this work, we identified seven genes in the dsa cluster including three regulatory genes (dsaA, dsaM, and dsaN), two transporter genes (dsaK and dsaL), and two other genes, dsaB (annotated as a phosphate synthase) and dsaJ (a PBP-type thioesterase). The DsaA and DsaN were unambiguously shown to be positive regulators of desotamide biosynthesis, and consistent with these roles, inactivation of either gene completely abolished desotamide production. Moreover, overexpression of dsaA or dsaN (independent of each other) was shown to improve desotamide titers. Production of desotamides in M1152/07-6H::dsaA strain was 2.4-fold greater than that in the heterologous dsa expression strain M1152/07-6H whereas desotamide titers from the M1152/07-6H::dsaN strain were about twice that of M1152/07-6H. In addition, inactivation of dsaB and dsaJ (independent of each other) completely abolished desotamide production, indicating their indispensability for desotamide assembly. These studies provide new insights into the functions and combinatorial biosynthetic potentials of seven key genes within the dsa biosynthetic gene cluster. Findings reported here are likely to facilitate further efforts aimed at assessing and developing the desotamides and related analogs for future applications.