Constitutive overexpression of asm18 increases the production and diversity of maytansinoids in Actinosynnema pretiosum
Constitutive overexpression of asm18 increases the production and diversity of maytansinoids in Actinosynnema pretiosum
复制标题
asm18 的组成型过表达增加了 Actinosynnema pretiosum 中美登木素生物碱的产量和多样性
DOI：
10.1007/s00253-015-7127-7
复制
发表时间：
2016-03-01
影响因子：
5
通讯作者：
Shen, Yuemao
中科院分区：
文献类型：
--
作者：
Li, Shanren;Lu, Chunhua;Shen, Yuemao
从放线菌综合征（Actinosynnema pretiosum）中分离到的Ansamitocins是一种有效的抗肿瘤化合物，属于美坦素家族，其抗体-美坦素偶联物目前正处于不同的临床试验阶段。抗菌菌素的临床应用刺激了广泛的研究，以提高其产量。在本研究中，我们研究了途径特异性S treptomyces抗生素调控蛋白（SARP）家族调控因子Asm18的功能，发现Asm18基因的异位过表达使HGF052 + pJTU824- Asm18菌株的n -去甲基-4,5-去环氧-maytansinol(2)的产量增加到50 mg/L，比对照菌株HGF052 + pJTU824增加4.7倍。Real-time PCR分析显示，asm18基因的过表达选择性地增加了起始单元（starter unit, asm43）、聚酮组装（polyketide assembly, asmA）、pks后修饰（after - pks modification, asm21）等生物合成相关基因的转录水平，以及调控基因（regulatory gene, asm8）的转录水平，asm8是抗氨霉素生物合成中特异性的lal型激活因子。随着发酵滴度的增加，从HGF052 + pJTU824-asm18菌株中分离到7个ansamitocin类似物（1-7），其中包括3个新类似物（1、5、6）和maytansinol(7)。我们的研究结果不仅为进一步提高抗炎菌素类似物的生产铺平了道路，而且表明抗炎菌素生物合成的pks后修饰是灵活的，通过优化HGF052和/或HGF052 + pJTU824-asm18菌株，这可能会产生maytansinol，这是合成抗体-maytansinoid偶联物的最有趣的中间体。
Ansamitocins isolated from Actinosynnema pretiosum, potent antitumor compounds, belong to the family of maytansinoids, and the antibody-maytansinoid conjugates are currently under different phases of clinical trials. The clinical applications of ansamitocins have stimulated extensive studies to improve their production yields. In this study, we investigated the function of a pathway-specific S treptomyces antibiotic regulatory protein (SARP) family regulator, Asm18, and observed that ectopic overexpression of the asm18 gene increased the production of N-demethyl-4,5-desepoxy-maytansinol (2) to 50 mg/L in the HGF052 + pJTU824-asm18 strain, an increase by 4.7-fold compared to that of the control strain HGF052 + pJTU824. Real-time PCR analysis showed that the overexpression of the asm18 gene selectively increased the transcription levels of the genes involved in the biosynthesis of the starter unit (asm43), polyketide assembly (asmA), post-PKS modification (asm21), as well as the transcription levels of the regulatory gene (asm8), which is a specific LAL-type activator in ansamitocin biosynthesis. With the increase of fermentation titre, seven ansamitocin analogs (1-7) including three new ones (1, 5, and 6) and maytansinol (7) were isolated from the HGF052 + pJTU824-asm18 strain. Our results not only pave the way for further improving the production of ansamitocin analogs but also indicate that the post-PKS modifications of ansamitocin biosynthesis are flexible, which brings a potential of producing maytansinol, the most fascinating intermediate for the synthesis of antibody-maytansinoid conjugates, by optimizing the HGF052 and/or HGF052 + pJTU824-asm18 strains.