Identifying Orphan Enzymes in Pseudomonas Putida Using Cobra-Based Methods

Genome-scale metabolic models have a wide variety of applications. While these models capture gene-protein-reaction relationships, they often are incomplete because they include misidentified reactions or metabolic gaps due to inaccurate genome annotations. Identifying genes responsible for missing reactions will provide a more complete understanding of metabolic networks and genotype-phenotype relationships. Previously, we developed a model-enabled gene search (MEGS) method to identify missing genes in a metabolic network associated with gap-filled reactions using functional selection experiments. MEGS involved creating a genomic library for a species with gap-filled reactions, and transforming the library into a recipient strain, such as Escherichia coli, whereby, the missing gap-filled reaction is coupled to growth [1]. Here, we extend MEGS to find genes associated with orphan enzymes in Pseudomonas putida. Databases exist that list orphan enzymes from many different organisms, whose metabolic activity has been shown experimentally but for which no associated gene sequences are known (including ~17% of enzymes with EC numbers) [2]. To verify some of the orphan enzymes in P. putida, we used COBRA methods to design E. coli recipient strains, which can only grow if they express genes associated with the appropriate orphan enzyme from P. putida. By finding sequences for orphan enzymes, we can build new GPR associations, thereby improving model predictions not only for P. putida but for other organisms with similar genes as well.

References:

1. Pan, S., Nikolakakis, K., Adamczyk, P. A., Pan, M., Ruby, E. G., & Reed, J. L. (2017). Model-enabled gene search (MEGS) allows fast and direct discovery of enzymatic and transport gene functions in the marine bacterium Vibrio fischeri. Journal of Biological Chemistry, 292(24), 10250–10261.
2. Shearer, A. G., Altman, T., & Rhee, C. D. (2014). Finding sequences for over 270 orphan enzymes. PLoS ONE, 9(5).