期刊
ACS SYNTHETIC BIOLOGY
卷 6, 期 1, 页码 39-44出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.6b00160
关键词
cell-free protein synthesis; natural products; diketopiperazine; cyclic dipeptide; biosynthesis; synthetic biology
资金
- Departments of Chemistry and Molecular Biosciences at Northwestern University [AT009143-13]
- David and Lucile Packard Foundation [2011-37152]
- ARPA-E [DE-AR0000435]
- Camille Dreyfus Teacher Scholar Award
- DARPA 1KM Program [HR0011-15-C-0084]
Genome sequencing has revealed that a far greater number of natural product biosynthetic pathways exist than there are known natural products. To access these molecules directly and deterministically, a new generation of heterologous expression methods is needed. Cell-free protein synthesis has not previously been used to study nonribosomal peptide biosynthesis, and provides a tunable platform with advantages over conventional methods for protein expression. Here, we demonstrate the use of cell-free protein synthesis to biosynthesize a cyclic dipeptide with correct absolute stereochemistry. From a single-pot reaction, we measured the expression of two nonribosomal peptide synthetases larger than 100 kDa, and detected high-level production of a diketopiperazine. Using quantitative LC-MS and synthetically prepared standard, we observed production of this metabolite at levels higher than previously reported from cell-based recombinant expression, approximately 12 mg/L. Overall, this work represents a first step to apply cell-free protein synthesis to discover and characterize new natural products.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据