title: In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis.
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.
authors.affiliation: Department of Molecular Biosciences, and the Feinberg School of Medicine, ‡Department of Chemistry, and §Department of Chemical and Biological Engineering, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Goering
authors.affiliation: Department of Molecular Biosciences, and the Feinberg School of Medicine, ‡Department of Chemistry, and §Department of Chemical and Biological Engineering, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Molecular Biosciences, and the Feinberg School of Medicine, ‡Department of Chemistry, and §Department of Chemical and Biological Engineering, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: McClure
authors.affiliation: Department of Molecular Biosciences, and the Feinberg School of Medicine, ‡Department of Chemistry, and §Department of Chemical and Biological Engineering, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Regan J
authors.initials: RJ
authors.lastname: Thomson
authors.affiliation: Department of Molecular Biosciences, and the Feinberg School of Medicine, ‡Department of Chemistry, and §Department of Chemical and Biological Engineering, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
authors.affiliation: Department of Molecular Biosciences, and the Feinberg School of Medicine, ‡Department of Chemistry, and §Department of Chemical and Biological Engineering, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D053898:Biosynthetic Pathways; D002474:Cell-Free System; D002853:Chromatography, Liquid; D004247:DNA / Q000235:genetics*; D004151:Dipeptides / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006096:Gramicidin / Q000096:biosynthesis / Q000737:chemistry; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D046908:Peptide Biosynthesis, Nucleic Acid-Independent / Q000235:genetics*; D010453:Peptide Synthases / Q000378:metabolism; D010456:Peptides, Cyclic / Q000096:biosynthesis / Q000737:chemistry; D010879:Piperazines / Q000737:chemistry / Q000378:metabolism; D058615:Synthetic Biology
chemical_list: D004151:Dipeptides; D010456:Peptides, Cyclic; D010879:Piperazines; D006096:Gramicidin; C032442:phenylalanyl-prolyl diketopiperazine; D004247:DNA; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: biosynthesis; cell-free protein synthesis; cyclic dipeptide; diketopiperazine; natural products; synthetic biology