Asm8, a specific LAL-type activator of 3-amino-5-hydroxybenzoate biosynthesis in ansamitocin production
Asm8, a specific LAL-type activator of 3-amino-5-hydroxybenzoate biosynthesis in ansamitocin production
复制标题
Asm8，安丝菌素生产中 3-氨基-5-羟基苯甲酸酯生物合成的特定 LAL 型激活剂
DOI：
10.1007/s11427-013-4502-4
复制
发表时间：
2013-07-01
影响因子：
9.1
通讯作者：
Deng ZiXin
中科院分区：
文献类型：
--
作者：
Pan WenQin;Kang QianJin;Deng ZiXin
高效抗肿瘤剂安丝菌素P3是一种分离自放射丝胞浆菌ATCC 31565的大环内酰胺。一个120 kb的DNA片段先前被鉴定为安丝菌素生物合成基因簇，并且包含用于聚酮化合物组装、前体合成、聚酮化合物合成后修饰和调控的基因。在生物合成基因簇中，asm 8编码一个1117个氨基酸的蛋白质，与大的ATP结合LuxR家族型调节因子具有高度相似性。在目前的研究中，我们确定了在ATCC 31565中通过基因置换使asm 8失活导致安丝菌素生产的完全丧失，并且与克隆的asm 8基因互补恢复了安丝菌素的生物合成。有趣的是，asm 8的破坏降低了负责3-氨基-5-羟基苯甲酸酯（AHBA）形成的基因的转录，AHBA是安丝菌素生物合成所需的起始单位。随后，将外源AHBA喂入asm 8突变体恢复了安丝菌素的生物合成，这表明Asm 8是AHBA生物合成的特异性正调控因子。此外，对asm 8同源物的研究鉴定了两个新的安丝菌素产生者，并在A. pretiosum ATCC 31280消除了该菌株中安丝菌素的产生。正调节剂Asm 8的表征和两种新安丝菌素生产者的发现为进一步提高这种重要抗肿瘤剂的生产铺平了道路。
The highly potent antitumor agent ansamitocin P3 is a macrolactam isolated from Actinosynnema pretiosum ATCC 31565. A 120-kb DNA fragment was previously identified as the ansamitocin biosynthetic gene cluster, and contains genes for polyketide assembly, precursor synthesis, post-polyketide synthesis modification, and regulation. Within the biosynthetic gene cluster, asm8 encodes an 1117-amino-acid protein with a high degree of similarity to the large ATP-binding LuxR family-type regulators. In the current study, we determined that inactivation of asm8 by gene replacement in ATCC 31565 resulted in the complete loss of ansamitocin production, and that complementation with a cloned asm8 gene restored ansamitocin biosynthesis. Interestingly, the disruption of asm8 decreased the transcription of genes responsible for 3-amino-5-hydroxybenzoate (AHBA) formation, the starter unit required for ansamitocin biosynthesis. Subsequently, feeding of exogenous AHBA to the asm8 mutant restored ansamitocin biosynthesis, which showed that Asm8 is a specific positive regulator in AHBA biosynthesis. In addition, investigation of asm8 homologs identified two new ansamitocin producers, and inactivation of the asm8 homolog in A. pretiosum ATCC 31280 abolished ansamitocin production in this strain. Characterization of the positive regulator Asm8 and discovery of the two new ansamitocin producers paves the way for further improving production of this important antitumor agent.