Enhancement of FK506 production through engineering the regulatory genes and precursor supply

FK506 (tacrolimus) is a 23-membered polyketide macrolide with immunosuppressant activity that is produced by a variety of Streptomyces species. Due to its pharmacological importance and broad applicability, considerable effort has been expended over the past few decades to elucidate its biosynthetic gene cluster and enhance the yield of FK506. In the study reported here, the level of FK506 production in S. clavuligerus CKD1119 and Streptomyces sp. Strain KCTC 11604BP was enhanced by promoting the intracellular pool of methylmalonyl-CoA and engineering the regulatory genes. The methylmalonyl-CoA mutase pathway participating in the biosynthesis of methylmalonyl-CoA from succinyl-CoA was introduced to the wild-type strain of S. clavuligerus CKD1119 along with the supplementation of methyl oleate to the medium, resulting in an approximately three fold improvement in the FK506 titer. Recently, the sequences of the entire FK506 biosynthetic gene clusters from Streptomyces sp. Strain KCTC 11604BP was reported and analyzed. Based on this, the tcs7 and fkbN genes encoded an LysR-type transcriptional regulator and LAL (large ATP-binding regulators of LuxR) family regulator, respectively. We discovered that fkbN overexpression in the tcs7 deletion strain resulted in a 4.0-fold (21 mg liter^-1) increase in FK506 production. These results implies that fkbN and tcs7 play essential roles as positive and negative regulators of FK506 biosynthetic pathway and demonstrate the potential of manipulate regulatory genes to increase the level of FK506 biosynthesis in industrial production strains.