FK506 is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces tsukubaensis. However, the low titer at which it is produced is a bottleneck to its application and use in industrial processes. We have overexpressed five potential targets associated with FK506 production (fkbO, fkbL, fkbP, fkbM, fkbD) which were identified in our previous study, with the aim to improve FK506 production. The results of the analysis showed that the constructed strains with an additional copy of each gene increased FK506 production by approximately 10-40 % compared with the wild-type strain D852. The results of the gene expression analysis indicated that each gene was upregulated. Combinatorial overexpression of the five genes resulted in a 146 % increase in the FK506 titer to 353.2 mg/L, in comparison with the titer produced by D852. To further improve the production of FK506 by the engineered strain HT-FKBOPLMD, we supplemented the medium with various nutrients, including soybean oil, lactate, succinate, shikimate, chorismate, lysine, pipecolate, isoleucine and valine. Optimization of feeding concentrations and times resulted in HT-FKBOPLMD being able to produce approximately 70 % more FK506, thereby reaching the maximal titer of 457.5 mg/L, with lower amounts of by-products (FK520 and 37,38-dihydro-FK506). These results demonstrate that the combination of the metabolically engineered secondary pathways and the exogenous feeding strategies developed here was able to be successfully applied to improve the production of industrially and clinically important compounds.