Division of Bioscience and Bioinformatics, Myongji University, Youngin, South Korea.
Appl Environ Microbiol. 2012 Apr;78(7):2249-55. doi: 10.1128/AEM.06766-11. Epub 2012 Jan 20.
FK506 is an important 23-member polyketide macrolide with immunosuppressant activity. Its entire biosynthetic gene cluster was previously cloned from Streptomyces sp. strain KCTC 11604BP, and sequence analysis identified three putative regulatory genes, tcs2, tcs7, and fkbN, which encode proteins with high similarity to the AsnC family transcriptional regulators, LysR-type transcriptional regulators, and LAL family transcriptional regulators, respectively. Overexpression and in-frame deletion of tcs2 did not affect the production of FK506 or co-occurring FK520 compared to results for the wild-type strain, suggesting that tcs2 is not involved in their biosynthesis. fkbN overexpression improved the levels of FK506 and FK520 production by approximately 2.0-fold, and a deletion of fkbN caused the complete loss of FK506 and FK520 production. Although the overexpression of tcs7 decreased the levels of FK506 and FK520 production slightly, a deletion of tcs7 caused 1.9-fold and 1.5-fold increases in FK506 and FK520 production, respectively. Finally, fkbN overexpression in the tcs7 deletion strain resulted in a 4.0-fold (21 mg liter(-1)) increase in FK506 production compared to that by the wild-type strain. This suggests that fkbN encodes a positive regulatory protein essential for FK506/FK520 biosynthesis and that the gene product of tcs7 negatively regulates their biosynthesis, demonstrating the potential of exploiting this information for strain improvement. Semiquantitative reverse transcription-PCR (RT-PCR) analyses of the transcription levels of the FK506 biosynthetic genes in the wild-type and mutant strains proved that most of the FK506 biosynthetic genes are regulated by fkbN in a positive manner and negatively by tcs7.
FK506 是一种具有免疫抑制活性的重要 23 元聚酮大环内酯。其完整的生物合成基因簇先前已从链霉菌属菌株 KCTC 11604BP 中克隆得到，序列分析鉴定出三个假定的调节基因 tcs2、tcs7 和 fkbN，它们分别编码与 AsnC 家族转录调节剂、LysR 型转录调节剂和 LAL 家族转录调节剂具有高度相似性的蛋白质。与野生型菌株相比，tcs2 的过表达和框内缺失都不会影响 FK506 或同时产生的 FK520 的产生，这表明 tcs2 不参与它们的生物合成。fkbN 的过表达使 FK506 和 FK520 的产量提高了约 2.0 倍，而 fkbN 的缺失则导致 FK506 和 FK520 的完全丧失。虽然 tcs7 的过表达略微降低了 FK506 和 FK520 的产量，但 tcs7 的缺失分别使 FK506 和 FK520 的产量增加了 1.9 倍和 1.5 倍。最后，fkbN 在 tcs7 缺失菌株中的过表达使 FK506 的产量与野生型菌株相比增加了 4.0 倍（21 毫克/升）。这表明 fkbN 编码一种对 FK506/FK520 生物合成至关重要的正调控蛋白，而 tcs7 的产物负调控它们的生物合成，这表明可以利用这些信息来进行菌株改良。野生型和突变菌株中 FK506 生物合成基因转录水平的半定量 RT-PCR（RT-PCR）分析证明，大多数 FK506 生物合成基因受 fkbN 的正向和 tcs7 的负向调控。
