Streptomyces genome analyses have revealed an enormous abundance of gene clusters codifying enzymes from secondary metabolism inferring that their potential to produce metabolites is underestimated. Among the mostly useful natural products, those biosynthesized by nonribosomal peptides synthetases share both high molecular complexity and therapeutic activity. A gene cluster encoding the biosynthesis of valinomycin, a depsipeptide ionophore, was annotated from the whole genome sequencing of the endophytic strain Streptomyces sp. CBMAI 2042 isolated from Citrus ssp. In the present study, we describe the association of fermentation process, molecular networking and mass spectrometry imaging which revealed the production of the ionophore by the endophytic strain. Additionally, the heterologous expression of the entire valinomycin gene cluster in S. coelicolor M1146 host was successfully promoted, representing an alternative and unique platform for production of new valinomycin derivatives in future studies.