This disclosure relates to validamycin A biosynthesis and in particular, to methods of producing validamycin A analogs and uses thereof. In a particular example, a method for making a validamycin A analog includes transforming a host cell with one or more recombinant DNA vectors to produce a valN-inactivated mutant; and culturing the valN-inactivated mutant in a culture medium to produce a validamycin A analog, such as 1,1'-bis-valienamine and validienamycin, and their conversion to valienamine. The present disclosure further relates to compositions including such compounds as well as methods of using the compositions, such as for antifungal agents.