Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes

The
rate at which genome sequencing data is accruing demands enhanced
methods for functional annotation and metabolism discovery. Solute
binding proteins (SBPs) facilitate the transport of the first reactant
in a metabolic pathway, thereby constraining the regions of chemical
space and the chemistries that must be considered for pathway reconstruction.
We describe high-throughput protein production and differential scanning
fluorimetry platforms, which enabled the screening of 158 SBPs against
a 189 component library specifically tailored for this class of proteins.
Like all screening efforts, this approach is limited by the practical
constraints imposed by construction of the library, i.e., we can study
only those metabolites that are known to exist and which can be made
in sufficient quantities for experimentation. To move beyond these
inherent limitations, we illustrate the promise of crystallographic-
and mass spectrometric-based approaches for the unbiased use of entire
metabolomes as screening libraries. Together, our approaches identified
40 new SBP ligands, generated experiment-based annotations for 2084
SBPs in 71 isofunctional clusters, and defined numerous metabolic
pathways, including novel catabolic pathways for the utilization of
ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an
integrated strategy for realizing the full value of amassing genome
sequence data.