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

Abstract

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

References

  • References39
  • Citations5

Mentioned in this Paper

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

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