FK506 is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces tsukubaensis. However, the production capacity of the strain is very low. To improve production, atmospheric and room temperature plasma (ARTP) mutagenesis was adopted to get the initial strains used in genome shuffling (GS). After three rounds of GS, S. tsukubaensis R3-C4 was the most productive strain, resulting in a FK506 concentration of 335 μg/mL, 2.6 times than that of the original wild-type strain. Moreover, exogenous DMSO 4% (v/v) addition could induce efflux of FK506 and increased FK506 production by 27.9% to 429 μg/mL. Finally, analyses of the differences in morphology, fermentation characteristics and specific gene expression levels between S. tsukubaensis R3-C4 and the wild-type strain revealed that R3-C4 strain: has hampered spore differentiation, thicker mycelia and more red pigment, which are likely related to the downregulation of bldD and cdgB expression. In addition, the expression levels of fkbO, fkbP, dahp, pccB and prpE all showed up-regulation at diverse degrees compared to the wild-type S. tsukubaensis. Overall, these results show that a combined approach involving classical random mutation and exogenous feeding can be applied to increase FK506 biosynthesis and may be applied also to the improvement of other important secondary metabolites.

中文翻译：

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结合大气和室温等离子体 (ARTP) 诱变、基因组改组和二甲基亚砜 (DMSO) 进料以提高 Streptomyces tsukubaensis 的 FK506 产量。

FK506 是由 Streptomyces tsukubaensis 产生的具有免疫抑制活性的临床上重要的大环聚酮化合物。然而，该菌株的生产能力非常低。为了提高产量，采用大气和室温等离子体（ARTP）诱变来获得用于基因组改组（GS）的初始菌株。三轮 GS 后，S. tsukubaensis R3-C4 是最高产菌株，导致 FK506 浓度为 335 μg/mL，是原始野生型菌株的 2.6 倍。此外，添加 4% (v/v) 的外源 DMSO 可诱导 FK506 流出，并将 FK506 产量提高 27.9% 至 429 μg/mL。最后，通过分析筑波沙门菌R3-C4与野生型菌株在形态、发酵特性和特定基因表达水平上的差异，发现R3-C4菌株：阻碍了孢子分化，菌丝体更厚，红色素更多，这可能与 bldD 和 cdgB 表达的下调有关。此外，fkbO、fkbP、dahp、pccB和prpE的表达水平与野生型S. tsukubaensis相比均呈现不同程度的上调。总体而言，这些结果表明，涉及经典随机突变和外源喂养的组合方法可用于增加 FK506 的生物合成，也可用于改善其他重要的次级代谢物。