Model-driven exploration of underground metabolism reveals drivers of metabolic innovation in Pseudomonas putida

Abstract

Beyond the heterologous expression of genes to generate microbes with novel properties, evolutionary engineering offers a complementary approach by exploiting adaptive processes to refine and expand cellular functionalities in biotechnology. A promising source for the generation of novel metabolic functions is the so-called underground metabolism, i.e., the subnetwork conformed by catalytically inefficient promiscuous enzymatic activities without an apparent physiological role. In this work, the potential of this underground metabolism as a source of novel phenotypes has been assessed in the soil bacterium Pseudomonas putida KT2440. To accomplish this, the high-quality genome-scale metabolic model iJN1462 was updated and expanded by the known set of promiscuous activities in this organism. The new metabolic space was explored to detect latent metabolic traits that could emerge through adaptive laboratory evolution (ALE) in order to broaden the range of available nutrients for P. putida. Using ALE, strains capable to degrade N-acetyl-L-alanine, an overproduced metabolite in HIV patients, were obtained. A multidisciplinary characterization of these evolved strains revealed that adaptation arose through synergistic and additive effects involving modifications in enzymes and transcription factors. This work demonstrates how underground metabolism can be exploited to expand the metabolic versatility of P. putida.