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.

FK506（他克莫司）是一种具有免疫抑制活性的23元大环内酯类抗生素，目前临床上广泛应用于骨髓、肝、肾移植。然而，FK506 的大规模工业应用由于其产量低而受到限制。在这项研究中，我们采用了一系列策略来提高 FK506 的产量。首先，通过 XylE 报告系统在S. tsukubaensis L19 中发现了一个强启动子gapdhp。然后在gapdhp的控制下，两个簇状调节剂（CSR）FkbN和Tcs7共同过表达产生菌株 L24，其 FK506 滴度从 171 mg/L 显着提高到 434 mg/L。用丙氨酸和异亮氨酸补充生长培养基进一步将产量提高到 493 mg/L 和 542 mg/L。接下来，通过 Plackett-Burman (PB) 设计确定培养基的显着成分，发现酵母提取物、棉籽粕和 KH ₂ PO _{4 的}最佳值为15.9 g/L、17.2 g/L 和 5.0 g/ L，分别。使用基于响应面方法 (RSM) 的 Box-Behnken (BB) 设计，开发了一个模型来预测最大效价为 747 mg/L。使用优化的培养基，菌株 L24 在 5 L 发酵罐中放大生产 FK506，在 192 小时发酵后达到 756 mg/L 的最大效价。