Reconstituted Biosynthesis of the Nonribosomal Macrolactone Antibiotic Valinomycin in Escherichia coli
Reconstituted Biosynthesis of the Nonribosomal Macrolactone Antibiotic Valinomycin in Escherichia coli
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DOI：
10.1021/sb400082j
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发表时间：
2014-07-01
影响因子：
4.7
通讯作者：
Neubauer, Peter
中科院分区：
文献类型：
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作者：
Jaitzig, Jennifer;Li, Jian;Neubauer, Peter
非核糖体肽（nrp）的结构复杂性阻碍了经济的化学合成和来源生物的可培养性差，限制了新型生物活性化合物的生物过程的发展。由于非核糖体肽合成酶（NRPSs）从简单的氨基酸构建块组装nrp，因此在一个强大且易于操作的表达宿主中异源表达NRPSs是一种有吸引力的策略，可以使药学上相关的nrp更容易获得，也是这些酶的工程设计以产生新的合成生物活性化合物的基础。在这里，我们展示了一种在大肠杆菌中以可溶性和活性形式表达来自tsusimaensis的654 kDa异二聚体valinomycin合成酶（VlmSyn）的系统方法。体外测定了VlmSyn活性和前体需求，并为先前提出的缬霉素生物合成模型提供了证据。重组valinomycin是一种具有抗真菌、抗菌和抗病毒活性的大内酯类抗生素，利用工程大肠杆菌菌株在廉价培养基中生长，无需补充前体，也无需进一步优化培养条件，就能在体内生产重组valinomycin。在大肠杆菌中剪裁VlmSyn为将来生产新的缬霉素类似物铺平了道路。
The structural complexity of nonribosomal peptides (NRPs) impeding economic chemical synthesis and poor cultivability of source organisms limits the development of bioprocesses for novel bioactive compounds. Since nonribosomal peptide synthetases (NRPSs) assemble NRPs from simple amino acid building blocks, heterologous expression of NRPSs in a robust and easy to manipulate expression host is an attractive strategy to make pharmaceutically relevant NRPs more accessible and is also a basis for engineering of these enzymes to generate novel synthetic bioactive compounds. Here we show a systematic approach for the heterologous expression of the 654 kDa heterodimeric valinomycin synthetase (VlmSyn) from Streptomyces tsusimaensis in a soluble and active form in Escherichia coli. VlmSyn activity and precursor requirements were determined in vitro and provided evidence for a previously proposed model of valinomycin biosynthesis. In vivo production of recombinant valinomycin, a macrolactone antibiotic with reported antifungal, antibacterial, and antiviral activities, was achieved using an engineered E. coli strain growing in inexpensive media and independent of the supplementation with precursors and further optimization of the cultivation conditions. Tailoring of VlmSyn in E. coli paves the way to the production of novel valinomycin analogues in the future.