Elevation of FK506 production by regulatory pathway engineering and medium optimization in Streptomyces tsukubaensis

FK506 (tacrolimus) is a 23-membered macrolide antibiotic with immunosuppressive activity, which is now widely used clinically in bone marrow, liver, and kidney transplantation. However, large-scale industrial use of FK506 is limited due to its low yield. In this study, we employed a series of strategies to enhance FK506 production. First, a strong promoter gapdhp was identified in S. tsukubaensis L19 through the XylE reporter system. Then two cluster-situated regulators (CSRs), FkbN and Tcs7, were co-overexpressed under the control of gapdhp to generate strain L24, which showed significant improvement in FK506 titer from 171 mg/L to 434 mg/L. Supplementing the growth medium with alanine and isoleucine further improved production to 493 mg/L and 542 mg/L, respectively. Next, the significant components of the medium were determined by Plackett-Burman (PB) design, with optimum values of yeast extract, cottonseed meal and KH2PO4 found to be 15.9 g/L, 17.2 g/L, and 5.0 g/L, respectively. Using Box-Behnken (BB) design based on response surface methodology (RSM), a model was developed to predict the maximum titer with 747 mg/L. With the optimized medium, scale-up production of FK506 by strain L24 in a 5 L fermenter reached a maximum titer of 756 mg/L after 192 h fermentation.