期刊
METABOLIC ENGINEERING
卷 60, 期 -, 页码 37-44出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2020.03.009
关键词
Valinomycin; Cell-free systems; In vitro biosynthesis; Nonribosomal peptide; Natural product; Synthetic biology
资金
- National Natural Science Foundation of China [31971348, 31800720]
- Natural Science Foundation of Shanghai [19ZR1477200]
- Shanghai Pujiang Program [18PJ1408000]
- ShanghaiTech University
- National Institutes of Health [1U19AI142780-01]
- David and Lucile Packard Foundation
- Camille Dreyfus Teacher-Scholar Program
Natural products are important because of their significant pharmaceutical properties such as antiviral, antimicrobial, and anticancer activity. Recent breakthroughs in DNA sequencing reveal that a great number of cryptic natural product biosynthetic gene clusters are encoded in microbial genomes, for example, those of Streptomyces species. However, it is still challenging to access compounds from these clusters because many source organisms are uncultivable or the genes are silent during laboratory cultivation. To address this challenge, we develop an efficient cell-free platform for the rapid, in vitro total biosynthesis of the nonribosomal peptide valinomycin as a model. We achieve this goal in two ways. First, we used a cell-free protein synthesis (CFPS) system to express the entire valinomycin biosynthetic gene cluster (> 19 kb) in a single-pot reaction, giving rise to approximately 37 mu g/L of valinomycin after optimization. Second, we coupled CFPS with cell-free metabolic engineering system by mixing two enzyme-enriched cell lysates to perform a two-stage biosynthesis. This strategy improved valinomycin production similar to 5000-fold to nearly 30 mg/L. We expect that cell-free biosynthetic systems will provide a new avenue to express, discover, and characterize natural product gene clusters of interest in vitro.
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