Site-directed modification of the adenylation domain of the fusaricidin nonribosomal peptide synthetase for enhanced production of fusaricidin analogs
Site-directed modification of the adenylation domain of the fusaricidin nonribosomal peptide synthetase for enhanced production of fusaricidin analogs
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DOI：
10.1007/s10529-012-0913-8
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发表时间：
2012-07-01
影响因子：
2.7
通讯作者：
Kim, Beom Seok
中科院分区：
文献类型：
--
作者：
Han, Jae Woo;Kim, Eun Young;Kim, Beom Seok
由多粘类芽孢杆菌DBB 1709产生的杀镰孢菌素是对真菌和革兰氏阳性菌具有活性的脂肽抗生素。杀镰孢菌素的环状六肽结构由杀镰孢菌素合成酶（一种非核糖体肽合成酶）合成。第三模块（FusA-A3）的腺苷酸化结构域可以募集l-Tyr、l-Val、l-Ile、l-allo-Ile或l-Phe，这使杀镰孢菌素结构多样化。由于掺入I-Phe的杀镰孢菌素类似物（LI-F07）比其他类似物表现出更有效的抗微生物活性，我们修饰了FusA-A3的底物结合口袋中的特异性赋予序列以指导LI-F07的增强生产。基于短杆菌肽S合成酶1和短杆菌肽合成酶1的腺苷酸化结构域的比较，这两个合成酶都主要激活l-Phe，使用定点诱变产生具有改变的FusA-A3的六个突变株。M3（I239 W，I299 V）、M5（I299 V，G322 A，V330 I）和M6（S239 W，I299 V，G322 A，V330 I）突变体比野生型菌株产生显著更多的LI-F07。
Fusaricidins produced by Paenibacillus polymyxa DBB1709 are lipopeptide antibiotics active against fungi and Gram-positive bacteria. The cyclic hexapeptide structures of fusaricidins are synthesized by fusaricidin synthetase, a non-ribosomal peptide synthetase. The adenylation domain of the third module (FusA-A3) can recruit l-Tyr, l-Val, l-Ile, l-allo-Ile, or l-Phe, which diversifies the fusaricidin structures. Since the l-Phe-incorporated fusaricidin analog (LI-F07) exhibits more potent antimicrobial activity than other analogs, we modified a specificity-conferring sequence in the substrate binding pocket of FusA-A3 to direct the enhanced production of LI-F07. Base on comparison to the adenylation domain of gramicidin S synthetase 1 and tyrocidine synthetase 1, both of which mainly activate l-Phe, six mutant strains with altered FusA-A3 were generated using site-directed mutagenesis. M3 (I239W, I299V), M5 (I299V, G322A, V330I), and M6 (S239W, I299V, G322A, V330I) mutants produced significantly more LI-F07 than the wild-type strain.