In vitro reconstruction of nonribosomal peptide biosynthesis directly from DNA using cell-free protein synthesis

Anthony W. Goering, Jian Li, Ryan A. McClure, Regan J. Thomson, Michael C. Jewett, Neil L. Kelleher*

*Corresponding author for this work

Research output: Contribution to journalArticle

39 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)39-44
Number of pages6
JournalACS synthetic biology
Volume6
Issue number1
DOIs
StatePublished - 2017

Keywords

  • Biosynthesis
  • Cell-Free Protein Synthesis
  • Cyclic Dipeptide
  • Diketopiperazine
  • Natural Products
  • Synthetic Biology

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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