Inhibitory cross-talk upon introduction of a new metabolic pathway into an existing metabolic network

Abstract

Evolution or engineering of novel metabolic pathways can endow microbes with new abilities to degrade anthropogenic pollutants or synthesize valuable chemicals. Most studies of the evolution of new pathways have focused on the origins and quality of function of the enzymes involved. However, there is an additional layer of complexity that has received less attention. Introduction of a novel pathway into an existing metabolic network can result in inhibitory cross-talk due to adventitious interactions between metabolites and macromolecules that have not previously encountered one another. Here, we report a thorough examination of inhibitory cross-talk between a novel metabolic pathway for synthesis of pyridoxal 5′-phosphate and the existing metabolic network of Escherichia coli. We demonstrate multiple problematic interactions, including (i) interference by metabolites in the novel pathway with metabolic processes in the existing network, (ii) interference by metabolites in the existing network with the function of the novel pathway, and (iii) diversion of metabolites from the novel pathway by promiscuous activities of enzymes in the existing metabolic network. Identification of the mechanisms of inhibitory cross-talk can reveal the types of adaptations that must occur to enhance the performance of a novel metabolic pathway as well as the fitness of the microbial host. These findings have important implications for evolutionary studies of the emergence of novel pathways in nature as well as genetic engineering of microbes for "green" manufacturing processes. molecular evolution | metabolic engineering | serendipitous pathway | synthetic biology | biodegradation

Structural and functional innovations in the real-time evolution of new (βα) 8 barrel enzymes

“…In addition, the latter part of the evolutionary trajectory might have been influenced by negative selection to lose HisA activity, and thus to prevent potential inhibition of TrpF activity when the HisA substrate ProFAR is also present in the cell. The potential for such inhibitory cross-talk to reduce fitness has been demonstrated in a similar case involving a serendipitous pathway for cofactor biosynthesis (22). Experiments to test the roles of epistasis and inhibitory cross-talk are ongoing in our laboratories.…”

Network-level architecture and the evolutionary potential of underground metabolism

“…We consider two alternative hypotheses to resolve this issue. First, underground reactions might interfere with existing processes (17) and are therefore disfavored by selection. Second, underground reactions might endow the cell with novel capabilities, but only under specific environmental conditions that the population has not regularly encountered during its evolutionary history.…”

“…To test whether underground reactions tend to introduce harmful metabolites, we focused on metabolite toxicity, which has been implicated in the interference between a novel pathway and native metabolism (17). Toxicity of metabolites, as measured by IC 50 values (half maximum inhibitory concentration), were predicted using a chemoinformatics tool trained on data measuring the susceptibility of E. coli against a diverse set of chemicals (18).…”

Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering

“…Engineering bacteria for the efficient production of a desired compound often fails due to inhibitory interactions caused by the introduction of the heterologous pathway into an existing metabolic network Kim & Copley, 2012;Pitera et al, 2007). Moreover, low production levels can result from the insufficient supply of the heterologous enzymes with precursors (Sandmann, 2002).…”

“…In many cases the development of a new bioprocess or improvement of existing biosynthesis strategies is hampered by lack of knowledge about each component of the host cell chassis (Lam et al, 2012). The introduction of a novel metabolic pathway into an existing metabolic network can lead to unpredictable interactions Kim & Copley, 2012). As these interactions can impair the fitness of the engineered strain, an ideal chassis would be a wellcharacterized cell harbouring only the minimal set of functions required to synthesize a product of interest.…”