Assessing the Combinatorial Potential of the RiPP
Cyanobactin <i>tru</i> Pathway
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Abstract
Ribosomally produced
natural products, the RiPPs, exhibit features
that are potentially useful in the creation of large chemical libraries
using simple mutagenesis. RiPPs are encoded on ribosomal precursor
peptides, but they are extensively posttranslationally modified, endowing
them with properties that are useful in drug discovery and biotechnology.
In order to determine which mutations are acceptable, strategies are
required to determine sequence selectivity independently of the context
of flanking amino acids. Here, we examined the absolute sequence selectivity
of the trunkamide cyanobactin pathway, <i>tru</i>. A series
of random double and quadruple simultaneous mutants were synthesized
and produced in <i>Escherichia coli</i>. Out of a total
of 763 mutated amino acids examined in 325 unique sequences, 323 amino
acids were successfully incorporated in 159 sequences, leading to
>300 new compounds. Rules for <i>tru</i> sequence selectivity
were determined, which will be useful for the design and synthesis
of combinatorial biosynthetic libraries. The results are also interpreted
in comparison to the known natural products of <i>tru</i> and <i>pat</i> cyanobactin pathways
