Minimal lactazole scaffold for in vitro thiopeptide bioengineering
Minimal lactazole scaffold for in vitro thiopeptide bioengineering
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DOI：
10.1038/s41467-020-16145-4
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发表时间：
2020-05-08
影响因子：
16.6
通讯作者：
Onaka, Hiroyasu
中科院分区：
文献类型：
--
作者：
Vinogradov, Alexander A.;Shimomura, Morito;Onaka, Hiroyasu
Lactazole A是乳系链霉菌的一种隐型硫肽，由9.8kb的紧凑生物合成基因簇编码。本研究通过将柔性体外翻译（FIT）系统与重组生产的内酰胺唑生物合成酶相结合，建立了内酰胺唑a体外生物合成平台，称为FIT- laz系统。对内酰胺唑生物合成的系统剖析揭示了生物合成酶对底物的显著耐受性，并导致了最小内酰胺唑支架的发展，这种结构只需要6个翻译后修饰就可以进行大环化。这种最小的硫肽与FIT-Laz的有效组装打开了不同的内酰胺唑类似物的10个连续突变，14至62个成员的大环，18个氨基酸长尾区域，以及含有非蛋白质氨基酸的杂化硫肽的途径。这项工作表明，最小的乳酸唑支架适用于广泛的生物工程，并为探索硫肽未开发的化学空间提供了可能性。Lactazole A是乳系链霉菌的一种硫肽，由9.8kb的紧凑生物合成基因簇编码。在这里，作者展示了一个通过结合灵活的体外翻译系统和重组产生的内酰胺唑生物合成酶来体外生物合成内酰胺唑a的平台。
Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8kb biosynthetic gene cluster. Here, we establish a platform for in vitro biosynthesis of lactazole A, referred to as the FIT-Laz system, via a combination of the flexible in vitro translation (FIT) system with recombinantly produced lactazole biosynthetic enzymes. Systematic dissection of lactazole biosynthesis reveals remarkable substrate tolerance of the biosynthetic enzymes and leads to the development of the minimal lactazole scaffold, a construct requiring only 6 post-translational modifications for macrocyclization. Efficient assembly of such minimal thiopeptides with FIT-Laz opens access to diverse lactazole analogs with 10 consecutive mutations, 14- to 62-membered macrocycles, and 18 amino acid-long tail regions, as well as to hybrid thiopeptides containing non-proteinogenic amino acids. This work suggests that the minimal lactazole scaffold is amenable to extensive bioengineering and opens possibilities to explore untapped chemical space of thiopeptides. Lactazole A is a thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8kb biosynthetic gene cluster. Here, the authors show a platform for in vitro biosynthesis of lactazole A via a combination of a flexible in vitro translation system with recombinantly produced lactazole biosynthetic enzymes.