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Minimal lactazole scaffold for in vitro thiopeptide bioengineering.


ABSTRACT: Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8?kb 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.

SUBMITTER: Vinogradov AA 

PROVIDER: S-EPMC7210931 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Minimal lactazole scaffold for in vitro thiopeptide bioengineering.

Vinogradov Alexander A AA   Shimomura Morito M   Goto Yuki Y   Ozaki Taro T   Asamizu Shumpei S   Sugai Yoshinori Y   Suga Hiroaki H   Onaka Hiroyasu H  

Nature communications 20200508 1


Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb 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 develo  ...[more]

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