Tacrolimus (also named FK506) is a macrolide immunosuppressant widely used in current clinics to prevent graft rejection, to treat atopic dermatitis and to aid insertion of coronary stents. Additionally, it has shown antiviral, anticancer, neuroprotective and neuroregenerative properties. The main producer of tacrolimus is Streptomyces tsukubaensis, an actinomycete isolated from a soil sample in Tsukuba (Japan) in 1984. The production of tacrolimus is expensive because of two reasons: a) low production titers of producing strains and b) fermentation byproducts (such as ascomycin -FK520-) that impede tacrolimus purification from culture broths. Therefore, a lot of efforts have been made by scientists in the last decades to increase tacrolimus production. The aim of this Collection is to introduce methodologies used to increase tacrolimus yields and to unravel the nutritional regulation of its biosynthesis. These include classical approaches, such as: culture media optimization, precursor feeding, and genetic engineering of the strains (both for primary metabolism genes and for tacrolimus biosynthetic cluster genes). Moreover, omic-approaches have also been performed, including transcriptomics, proteomics, metabolomics, and bioinformatic metabolic modeling. This Collection also aims to introduce basic methodologies for the growth and genetic engineering of Streptomyces tsukubaensis, in order to share normalized protocols with the scientific community that could be also applied to other Streptomyces species.