Aug 2, 2016

In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

ACS Synthetic Biology
Anthony W GoeringNeil L Kelleher


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.

  • References39
  • Citations5


  • References39
  • Citations5

Mentioned in this Paper

Molecular Stereochemistry
Gramicidin P
Cyclic Peptides
Alkalescens-Dispar Group
Natural Products
Cyclic dipeptide oxidase
Reconstructive Surgical Procedures
phenylalanyl-prolyl diketopiperazine

Related Feeds

Artificial Cell Models

This feed focuses on biomimetrics, synthetic biology and bio- and tissue-engineering approaches used for modeling human diseases.

Biomaterial Engineering

Advances in biomaterial engineering have permitted the development of sophisticated drug-releasing materials with a biomimetic 3D support that allow a better control of the microenvironment of transplanted cells. Here is the latest research.

CZI Human Cell Atlas Seed Network

The aim of the Human Cell Atlas (HCA) is to build reference maps of all human cells in order to enhance our understanding of health and disease. The Seed Networks for the HCA project aims to bring together collaborators with different areas of expertise in order to facilitate the development of the HCA. Find the latest research from members of the HCA Seed Networks here.