The actinobacteria Actinomadura hibisca synthesizes the natural products pradimicin A-C through a type II polyketide biosynthetic pathway. Eight tailoring enzymes in pradimicin biosynthesis have been investigated in this work, including PdmJ, PdmW, PdmN, PdmT, PdmO, PdmS, PdmQ and PdmF. PdmJ and PdmW were characterized as cytochrome P450 hydroxylases that catalyze the incorporation of two hydroxyl groups at C-5 and C-6, respectively. These enzymes worked synergistically and their co-expression significantly improved the efficiency of the hydroxylation steps. PdmN is an amino acid ligase that accepts a variety of substrates and ligates a D-alanine moiety to C-16 to form the corresponding derivatives. PdmS and PdmQ were functionally identified as Oglycosyltransferases. Disruption of pdmS in the genome of A. hibisca generated a biosynthetic precursor without sugar moieties, which validated that PdmS is the first glycosyltransferase that attaches the first sugar to the 5-OH of pradimicins. In contrast, disruption of pdmQ led to the synthesis of pradimicin B, confirming that PdmQ was responsible for attaching the D-xylose moiety to the 3'-OH of the first sugar portion in pradimicins. Naturally, the first sugar moiety 4',6'-dideoxy-4'-amino-D-galactose of pradimicin A and B is methylated at 2'-NH. When the expression of PdmO was compromised, the mutant strain produced mainly pradimicin C, which contains the 4',6'- dideoxy-4'-amino-D-galactose in its structure. This suggested that PdmO was responsible for the N-methylation of the amino sugar. PdmF was identified as the C-11 Omethyltransferase. Moreover, PdmT was confirmed to be an O-methyltransferase through gene disruption and in vitro biochemical reactions. PdmT methylates the 7-OH to form a methoxy group that in a later step is removed to generate the pradimicin aglycon. In summary, this research has identified eight important pradimicin biosynthetic enzymes that are involved in various tailoring steps in pradimicin biosynthesis. Several new pradimicin analogues has been generated by manipulating these enzymes. Their enzymatic properties and collaborative actions were investigated. These results not only provide new insights into type II polyketide biosynthetic pathways, but also enable rational engineering of the pradimicin biosynthetic pathway to create new analogues for drug development.