Ribosomal route to small-molecule diversity - PubMed (original) (raw)

. 2012 Jan 11;134(1):418-25.

doi: 10.1021/ja208278k. Epub 2011 Dec 22.

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Ribosomal route to small-molecule diversity

Ma Diarey B Tianero et al. J Am Chem Soc. 2012.

Abstract

The cyanobactin ribosomal peptide (RP) natural product pathway was manipulated to incorporate multiple tandem mutations and non-proteinogenic amino acids, using eight heterologous components simultaneously expressed in Escherichia coli . These studies reveal the potential of RPs for the rational synthesis of complex, new small molecules over multiple-step biosynthetic pathways using simple genetic engineering.

© 2011 American Chemical Society

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Figures

Figure 1

Figure 1. Biosynthesis of tru pathway derivatives in E. coli

Numbered components indicate eight individual elements including enzymes, tRNA, or precursor peptide (TruE variants) that are required for synthesis of mature natural products. The orthologous tRNA and amino acyl tRNA synthetase components required to introduce non-proteinogenic amino acids are shown in blue. Enzymes from the tru pathway are indicated in orange. The precursor peptide TruE is shown with a helical, 35-amino acid leader sequence indicated schematically. X indicates any amino acid.

Figure 2

Figure 2. Expression strategy

Enzymes and two control molecules are synthesized constitutively from the vector ptru-SD1 (top). An internal control sequence and a variable region, that can lead to peptide libraries, is synthesized from ptruE (bottom). Optionally, non-proteinogenic amino acids can be included using a third vector, pEVOL (right).

Figure 3

Figure 3. Expression of mutant cyanobactins

E. coli cultures expressing 20 (left) and 15 (right) were harvested and analyzed by FT-MS. Extracted ion chromatograms showed that each culture expressed control compounds 3 (A) and 1 (B). In cells containing plasmids encoding 20, compound 20 could be detected (C), but not 15 (D), nor any other cyanobactin 4–22. Similarly, in cells encoding 15, only compound 15 (D) but not 20 (C), nor any other cyanobactin, could be detected. Thus, each experiment contained two internal positive controls and 19 external negative controls.

Figure 4

Figure 4. Optimization of non-proteinogenic amino acid incorporation

Compound 10 was synthesized in E. coli and analyzed by LC-ESI-MS. Optimum production was achieved in the absence of inducer (arabinose), whereas higher concentrations of inducer completely repressed synthesis. Two peaks are present because multiple stereoisomers are present (the a-proton adjacent to thiazoline is labile, and Pro undergoes cis-trans isomerization in this family).

Figure 5

Figure 5. Compounds synthesized in this study

Yellow indicates wild-type compounds, while pink bubbles indicate mutations that deviate from wild type. Hexa- (left), hepta- (mid) and octa- (right) peptide derivatives were synthesized using these methods. * indicates compounds for which only mono-prenylated (9, 11, and 12) or non-prenylated (18) derivatives were identified. For all other compounds, both singly, doubly, and sometimes triply prenylated products were identified.

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