An Integrated Approach to Discover and Bioengineer Bacterial Natural Products
Charles E. Melançon III
Department of Chemistry and Chemical Biology, University of New Mexico
Natural products have been honed by evolution to be bioactive, making them
excellent drugs and drug leads. The more than 20,000 bacterial genomes sequenced to
date provide us with the genetic blueprints for the synthesis of thousands of unstudied
natural products. However, the ability to systematically search these genomes for gene
clusters encoding structurally novel natural products and new biosynthetic enzyme
activities are still in their infancy.
To expand current capabilities, we have developed and used the software
package Dynamite to globally identify and annotate polyketide and non-ribosomal
peptide biosynthetic gene clusters in sequenced, publicly available genomes; and to
identify and classify conserved groups of homologous proteins within these gene
clusters. By integrating Dynamite data with experimental results from literature and 16S
phylogenetic data, we are able to more accurately predict structures of unstudied
natural products encoded in bacterial genomes and assess their structural novelty; to
identify unstudied natural product biosynthetic enzyme families; and to profile the
phylogenetic distribution of natural products in the bacterial kingdom to guide
bioprospecting.
Our current efforts focus on the biosynthetic pathways of bacterial type II
polyketides (PK-IIs). We are using our bioinformatic/phylogenetic data to guide
experimental efforts to 1) identify new PK-II natural products from rare Actinobacteria, 2)
select new organisms harboring PK-II gene clusters for next generation genome
sequencing, 3) construct refactored operons to produce and derivatize PK-II core
structures heterologously in Streptomyces, and 4) systematically profile Actinobacteria
from culture collections and the environment for novel PK-II biosynthetic genes.
We also have two ongoing bioengineering projects. In the first, we have
constructed engineered E. coli strains harboring an orthogonal ribosome-controlled
fluorescent reporter that are able to detect ribosome inhibition by a variety of
aminoglycosides in a highly sensitive, dose-dependent manner. Dose response patterns
observed for different aminoglycosides are consistent with current knowledge regarding
their potencies and mechanisms of action, suggesting that the system can be used to
rapidly assess target sites and relative potencies of ribosome inhibitors and to
investigate their mechanisms of action. In the second project, we have created an
amber suppression-based site-specific unnatural amino acid (UAA) incorporation
system for Streptomyces and have successfully incorporated p-iodophenylalanine and
the photocrosslinking/clickable UAA p-azidophenylalanine into a model protein in
Streptomyces venezuelae. We will apply this technology to probe protein-protein
interactions in natural product biosynthetic enzyme complexes.